Site-specific chemoenzymatic protein modifications

ABSTRACT

The present invention relates to methods and reagents for use in site-selective modification of proteins having lysine residues with functionalized peptides using a chemoenzymatic microbial transglutaminase-mediated reaction. The functionalized proteins may be used for study or therapeutic uses.

FIELD OF THE INVENTION

The present invention relates generally to a novel method of introducingmodifying groups to a protein. In particular, the present inventionrelates to the selective derivation of lysine residues in proteins usinga chemoenzymatic microbial transglutaminase-mediated reaction formodifying proteins and methods for their preparation and use.

BACKGROUND

It is well-known that the properties and characteristics of proteins maybe modified by conjugating groups to the protein. For example, U.S. Pat.No. 4,179,337 disclosed proteins conjugated to polyethylene orpolypropylene glycols. Generally, such conjugation generally requiressome functional group in the protein to react with another functionalgroup in a conjugating group. Amino groups, such as the N-terminal aminogroup or the ε-amino group in lysine residues have been used incombination with suitable acylating reagents for this purpose. It isoften desired or necessary to control the conjugation reaction, such aswhere the conjugating compounds are attached to the protein and tocontrol how many conjugating groups are attached. This is often referredto as specificity or selectivity.

Site-specific modification of proteins is a longstanding challenge inthe pharmaceutical and biotechnology arts. The classic methodsoftentimes lead to non-specific labeling (e.g. NHS Lys labeling) orrequire engineering (e.g. maleimide Cys labeling or unnatural aminoacids). In addition, the repertoire of selective chemical reactions,however, is very limited. One alternative is, by recombinant methods, tointroduce special unnatural amino acids having a unique reactivity andthen exploit this reactivity in the further derivatization. Anotheralternative is the use of enzymes which recognize structural andfunctional features of the protein to be modified. An example of this isthe use of microbial transglutaminase (mTGase) to selectively modify Glnresidues in growth hormone. Other documents disclose the use oftransglutaminase to alter the properties of physiologically activeproteins. See e.g. EP 950 665, EP 785 276 and Sato, Adv. Drug DeliveryRev., 54, 487-504 (2002), which disclose the direct reaction betweenproteins comprising at least one Gln and amine-functionalized PEG orsimilar ligands in the presence of transglutaminase; see also Wada inBiotech. Lett., 23, 1367-1372 (2001), which discloses the directconjugation of P-lactoglobulin with fatty acids by means oftransglutaminase. The reaction catalyzed by the transglutaminase is atransamidation reaction in which the primary amide of the glutamineresidue is converted to a secondary amide from a primary amine presentin the reaction mixture.

The selective derivatization of proteins remains a very difficult task;the derivatization of lysines in a protein by acylation is an even moreinherently non-selective process. Thus, there is at present no efficientmethod for the selective derivatization of lysine residues. Accordingly,there is a need in the art for methods of selectively derivatizing aminoacid residues such as lysine in proteins or polypeptides.

SUMMARY

In one aspect, a method for modifying a protein is disclosed. The methodpermits site selective modifications. The method includes providing atarget protein having at least one lysine residue; contacting the targetprotein with a modifying compound having the formulaR¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) in the presence of a microbialtransglutaminase to form a modified protein;

wherein x is 0 or 1; y is 0 or 1; z is 0 or 1;R¹ is selected from the group consisting of: acetyl,

wherein each R⁴ is selected from —H, —N₃, and

W is selected from: C₁-C₆ linear or branched alkyl or polyethyleneglycol having a molecular weight of between about 40 and about 80,000amu;A is absent or selected from —O—, —NH—, and —S—;B is absent or selected from —O—, —C(O)—, —NH—, —C(O)NH—, —NHC(O)—,—NHC(O)O—, —OC(O)NH—, —OC(O)O—, —C═N(OH)—, —S(O₂)—, —NHS(O₂)—,—S(O₂)NH—, —S(O)—, —NHS(O)—, —S(O)NH—; —C(O)O—, —OC(O)—, —S—, ═NH—O—,═NH—NH— and ═NH—N(C₁-C₂₀alkyl)-;R² is selected from the group consisting of: a fatty acid, linear orbranched C₁-C₃ alkyl-N₃, cyclooctynyl, fluorophore, polysaccharide,—CH(OCH₃)₂,

each n is an integer independently selected from 0 to 6;each Q is selected from H and —NO₂.

In some embodiments, the method also includes controlling the pHenvironment of the target protein to a pH greater than 7; and contactingthe modified protein with a molecule having a cysteine residue.

In some embodiments, the molecule having cysteine residue isN⁵—((R)-1-((carboxymethyl)amino)-3-mercapto-1-oxopropan-2-yl)-L-glutamine.

In some embodiments, microbial transglutaminase is Ajinomoto microbialtransgluaminase TI. In some embodiments, the protein is a carrierprotein. In some embodiments, the protein is CRM₁₉₇. In someembodiments, the protein is selected from: bacterial toxin, bacterialtoxin fragments, detoxified bacterial toxins, antibodies, and antibodyfragments.

In some embodiments, the method includes reacting the R₁ group with abiointeractive agent or an analytical agent.

In some embodiments, the method includes reacting the R² group with abiointeractive agent or an analytical agent. In some embodiments, theanalytical agent is a label. In some embodiments, the method includesdetecting the label.

In another aspect, conjugate prepared by the disclosed processes arealso disclosed. In another aspect, therapeutic proteins are disclosedprepared by the disclosed processes. In another aspect, imaging agentsare disclosed prepared by the disclosed processes. In another aspect,labelling tools are disclosed prepared by the disclosed processes.

In another aspect, compounds of the formulaR¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) are disclosed where R¹, x, y,A, W, B, R² and z have the meanings described herein.

In one aspect, a method for modifying a protein is disclosed. The methodincludes providing a target protein having at least one lysine residue;contacting the target protein with a modifying compound having theformula R¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z) in the presence of amicrobial transglutaminase, wherein x is 0 or 1; y is 0 or 1; z is 0 or1; R¹ is selected from the group consisting of: acetyl,

W is selected from C₁-C₆ linear or branched alkyl or polyethylene glycolhaving a molecular weight of between about 40 and about 80,000 amu;R² is selected from the group consisting of: linear or branched C₁-C₃alkyl-N₃, cyclooctynyl, fluorophore, polysaccharide,

to form a modified protein.

In some embodiments, x is 1. In some embodiments, R¹ is selected fromacetyl,

In some embodiments, R¹ is acetyl. In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, y is 1 and z is 0. In some embodiments, wherein yis 1 and z is 1.

In some embodiments, W is selected from C₁-C₆ linear or branched alkyl.In some embodiments, W is C₂ linear alkyl. In some embodiments, R² is

In some embodiments, x is 0, y is 1, and R¹ is

In some embodiments, wherein x is 0 and R¹ is

In some embodiments, y is 0 and z is 1. In some embodiments, y is 1 andz is 1.

In some embodiments, W is C₁-C₆ linear or branched alkyl. In someembodiments, W is C₂ linear alkyl. In some embodiments, W is C₅ linearalkyl. In some embodiments, W is linear or branched polyethylene glycolhaving a molecular weight of between about 40 and about 3000 amu. Insome embodiments, W is linear polyethylene glycol having a molecularweight of between about 40 and about 80 amu.

In some embodiments, R² is linear or branched C₁-C₃ alkyl-N₃. In someembodiments, R² is C₂-alkyl-N₃. In some embodiments, R² is cyclooctynyl.In some embodiments, R² is

In some embodiments, R² is

In some embodiments, R² is a fluorophore. In some embodiments, theflurophore is selected from: alexa 647, alexa 750, alexa 488, Cy5, Cy7,rhodamine, and fluorescein. In some embodiments the fluorophore is ofthe formula

where n is from 1 to 3 and each m is from 1 to 2. In some embodiments, nis 1. In some embodiments, n is 2. In some embodiments, n is 3. In someembodiments, one m is 1 and the other m is 2. In some embodiments, bothm's are 1. In some embodiments, both m's are 2. In some embodiments, R²is

In some embodiments, R² is a polysaccharide. In some embodiments, thepolysaccharide is selected from GBSII, GBSV, and MenA. In someembodiments, R² is GBSII. In some embodiments, R² is GBSV. In someembodiments, R² is MenA.

In some embodiments, the method includes controlling the pH environmentof the target protein to a pH greater than 7; and contacting themodified protein with a molecule having a cysteine residue. In someembodiments, the molecule having a cysteine residue isN⁵—((R)-1-((carboxymethyl)amino)-3-mercapto-1-oxopropan-2-yl)-L-glutamine.

In some embodiments, the microbial transglutaminase is Ajinomotomicrobial transgluaminase TI.

In some embodiments, the protein is a carrier protein. In someembodiments, the protein is CRM₁₉₇. In some embodiments, the protein isselected from: bacterial toxin, bacterial toxin fragments, detoxifiedbacterial toxins, antibodies, and antibody fragments.

In some embodiments, the method also includes reacting the R₁ group witha biointeractive agent or an analytical agent. In some embodiments, themethod includes reacting the R² group with a biointeractive agent or ananalytical agent. In some embodiments, the analytical agent is a label.In some embodiments, the method also includes detecting the label.

In another aspect, a conjugate is disclosed that is prepared from themethods disclosed herein. In another aspect, a vaccine is disclosed thatis prepared with a conjugate or a modified protein disclosed herein. Inanother aspect, a therapeutic protein is disclosed having a modifiedprotein disclosed herein. In another aspect, an imaging agent isdisclosed having a modified protein disclosed herein. In another aspect,a labeling tool is disclosed having modified protein disclosed herein.

In another aspect, compound of the formulaR¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z) are disclosed wherein x is 0 or1; y is 0 or 1; z is 0 or 1; R¹ is selected from the group consistingof: acetyl,

W is selected from C₁-C₆ linear or branched alkyl or polyethylene glycolhaving a molecular weight of between about 40 and about 80,000 amu; R²is selected from the group consisting of: linear or branched C₁-C₃alkyl-N₃, cyclooctynyl, fluorphore, polysaccharide,

In some embodiments, the compound is selected from the group consistingof:

In some embodiments, a conjugate of a compound is disclosed wherein theconjugate includes a conjugate protein and a compound disclosed herein.In some embodiments, the conjugate protein is CRM₁₉₇. In someembodiments, the conjugate protein is GBS₈₀.

In some embodiments, a vaccine is disclosed comprising a conjugatedisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a synthetic scheme depicts the addition of a protein modifyinggroup reacting with a protein (CRM197) catalyzed by microbialtransglutaminase.

FIG. 2 is an SDS-Page gel electrophoresis characterizing the conjugationof MenA polysaccharide with CRM₁₉₇ using a compound of the invention.

FIG. 3 is an SDS-Page gel electrophoresis characterizing the siteselective conjugation of GBS80 antigenic polysaccharide with CRM₁₉₇using a compound of the invention, wherein lane 1 is MW, lane 2 isGBS80-K—N₃, lane 3 is GBS80-K—N₃/PSV 1 mg of protein, lane 4 isGBS80-K—N₃/PSV 1 mg of protein, and lane 5 is GBS80-K—N₃/PSV 1 mg ofprotein.

FIG. 4 is an SDS-Page gel electrophoresis characterizing the resultingreaction of a conjugated GBS80 antigenic polysaccharide with CRM₁₉₇using a compound of the invention, wherein lane 1 is MW, lane 2 isGBS80-K—N₃, lane 3 is GBS80-K—N₃/PSII 1 mg of protein, lane 4 isGBS80-K—N₃/PSII 1 mg of protein, and lane 5 is GBS80-K—N₃/PSII 1 mg ofprotein.

FIG. 5 shows ELISA immunoassay results for determination of Ig titersagainst GBS II polysaccharide antigen, wherein ELISA anti PSII IgG andsurvival results at 1.0 ug dose of PS.

FIG. 6 shows ELISA immunoassay results for determination of Ig titersagainst GBS V polysaccharide antigen, wherein ELISA anti PSV IgG andsurvival results at 1.0 ug dose of PS.

FIG. 7 shows opsonophagocytosis assay results for using GBS strains.

DETAILED DESCRIPTION

All references made to patents, patent publications, and otherliterature are made for their incorporation into this disclosure to theextent permissible by law.

The present invention addresses the aforementioned needs by providing amethod of introducing modifying compounds to a target protein in aselective manner via reaction with a modifying compound, while usingconventional chemical methods. The method is generally depicted inFIG. 1. A lysine residue from a target protein (for example CRM₁₉₇)reacts with a glutamine residue (Gln) from a modifying compound offormula (I): R¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) or of formula(II): R¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z). The resulting product isa protein having one or more groups capable of further chemicalfunctionalization.

In one aspect, a process for modifying a protein includes: (a) formingan activated complex between an auxiliary protein and a modifyingcompound by catalytic action of microbial transglutaminase; (b)transferring the modifying compound from the activated complex to atarget protein thereby creating a modified protein. As such, a “modifiedprotein” as used herein, refers to a protein or polypeptide that hasbeen selectively modified by addition of a modifying compound usingmicrobial transglutaminase.

In some embodiments, the method includes a transglutaminase catalyzedreaction of a target protein having at least two lysines residues with amodifying compound. The modifying compound is a glutamine-containingprotein of the formula (I): R¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) orof formula (II): R¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z).

In this process, an activated acyl complex is formed by reacting theglutamine residue in the modifying compound with microbialtransglutaminase in order to attach the modifying compound. In oneembodiment, the modifying compound is transferred by acylation to alysine residue in the target protein. In one embodiment, R¹ and R² aredesired substituents, where at least one of them has a chemical groupthat is suitable for further modification. Thus, the process involves amicrobial transglutaminase reaction in order to selectively modify alysine residue in a target protein.

As used herein, the term “amu” is an abbreviation for atomic mass unitsalso frequently referred to as Dalton units.

As used herein, the term “polypeptide” refers to a polymer of amino acidresidues joined by peptide bonds, whether produced naturally orsynthetically. Polypeptides of less than about 10 amino acid residuesare commonly referred to as “peptides.” The term “peptide” is intendedto indicate a sequence of two or more amino acids joined by peptidebonds, wherein said amino acids may be natural or unnatural. The termencompasses the terms polypeptides and proteins, which may consist oftwo or more peptides held together by covalent interactions, such as forinstance cysteine bridges, or non-covalent interactions.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other nonpeptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless. A protein or polypeptide encoded by a non-host DNA moleculeis a “heterologous” protein or polypeptide.

An “isolated polypeptide” is a polypeptide that is essentially free fromcellular components, such as carbohydrate, lipid, or other proteinaceousimpurities associated with the polypeptide in nature. Typically, apreparation of isolated polypeptide contains the polypeptide in a highlypurified form, i.e., at least about 80% pure, at least about 90% pure,at least about 95% pure, greater than 95% pure, such as 96%, 97%, or 98%or more pure, or greater than 99% pure. One way to show that aparticular protein preparation contains an isolated polypeptide is bythe appearance of a single band following sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis of the protein preparation andCoomassie Brilliant Blue staining of the gel. However, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

A “biointeractive agent” as used herein refers to an organic moiety orcompound that invokes a biological response when introduced into aliving tissue or cell. Example of biointeractive agents includeantigens, toxins, therapeutic proteins and the like. Biointeractiveagents may be small molecules and macro molecules.

An “analytical agent” as used herein refers to an organic moiety orcompound that can be detected by instrumental methods for qualitativelyor quantitatively characterizing the material to which the analyticalagent is bound or otherwise associated. Examples of such analyticalagents include labels for example fluorophores or radio labels.

As used herein, the term “alkyl” is a C₁-C₄₅ alkyl group which is linearor branched. In some embodiments, alkyl is from C₁-C₂₀. In someembodiments, alkyl is from C₁-C₁₂. In some embodiments, alkyl is fromC₁-C₆. Where alkyl is defined as a group, such as W in formulas I and IIdiscussed herein, it should be understood that the group may also beknown as alkylene such that there is one substitution with an adjoininggroup or two substitutions between two adjoining groups.

As used herein, the term “polyethylene glycol” or “PEG” refers to apolyether compound with a repeating (O—CH₂—CH₂)_(n) subunit having amolecular weight of between about 40 and about 80,000 amu where n is aninteger representing the number of repeated ether subunits. Wherepolyethylene glycol is defined as a group, such as W in formulas I andII discussed herein, it should be understood that there is onesubstitution with an adjoining group in which case there may be a freealcohol group at a terminus or two substitutions between two adjoininggroups.

Transglutaminase

As mentioned above, a catalyst must be used for covalently linking themodifying compound to the target protein. The catalyst must be amicrobial transglutaminase (also interchangeably referred to herein as“mTGase”). The catalyst is also known asprotein-glutamine-γ-glutamyltransferase from microbial sources andcatalyzes the acyl transfer reaction between the γ-carboxyamido group ofa glutamine (Gln) residue in protein or a protein chain and the ε-aminogroup of a lysine (Lys) residue or various alkylamines.

The transglutaminase to be used in the methods of the present inventioncan be obtained from various microbial origins with no particularlimitation. Examples of useful microbial transglutaminases includetransglutaminases, such as from Streptomyces mobaraense, Streptomycescinnamoneum, and Streptomyces griseocarneum (all disclosed in U.S. Pat.No. 5,156,956, which is incorporated herein by reference), andStreptomyces lavendulae (disclosed in U.S. Pat. No. 5,252,469, which isincorporated herein by reference) and Streptomyces ladakanum(JP2003199569, which is incorporated herein by reference). It should benoted that members of the former genus Streptoverticillium are nowincluded in the genus Streptomyces [Kaempfer, J. Gen. Microbiol., 137,1831-1892, 1991]. Other useful microbial transglutaminases have beenisolated from Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183,which is incorporated herein by reference) and from various Myxomycetes.Other examples of useful microbial transglutaminases are those disclosedin WO 96/06931 (e.g. transglutaminase from Bacilus lydicus) and WO96/22366, both of which are incorporated herein by reference.

Modifying Compounds

Modifying compounds that may be used in the disclosed methods areglutamine-containing proteins of the general formulas: (I):R¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) or of formula (II):R¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z) where Leu refers to the aminoacid leucine (for example L-leucine) that is either present or absent(i.e. when x is 1 or 0, respectively); Gln refers to the amino acidglutamine (for example L-glutamine); Gly refers to the amino acidresidue glycine (for example L-glycine) that is either present or absent(i.e. when y is 1 or 0, respectively).

In some embodiments, x is 0. In some embodiments, x is 1. In someembodiments, y is 0. In some embodiments y is 1. In some embodiments, zis 0. In some embodiments, z is 1.

In some embodiments, R¹ is acetyl. In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, Q is H. In some embodiments, Q is —NO₂. In someembodiments, n is 0. In some embodiments, n is 1. In some embodiments, nis 2. In some embodiments, n is 3. In some embodiments, n is 4. In someembodiments, n is 5. In some embodiments, n is 6. In some embodiments,R⁴ is H. In some embodiments, R⁴ is —N₃. In some embodiments, R⁴ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, W is C₁-C₆ linear or branched alkyl. In someembodiments, W is polyethylene glycol having a molecular weight ofbetween about 40 and about 80,000 amu.

In some embodiments, A is absent. In some embodiments, A is —O—. In someembodiments, A is —NH—. In some embodiments, A is —S—.

In some embodiments, B is absent. In some embodiments, B is —O—. In someembodiments, B is —C(O)—. In some embodiments, B is —NH—. In someembodiments, B is —C(O)NH—. In some embodiments, B is —NHC(O)—. In someembodiments, B is, —NHC(O)O—. In some embodiments, B is —OC(O)NH—. Insome embodiments, B is —OC(O)O—. In some embodiments, B is —C═N(OH)—. Insome embodiments, B is —S(O₂)—. In some embodiments, B is —NHS(O₂)—. Insome embodiments, B is —S(O₂)NH—. In some embodiments, B is —S(O)—. Insome embodiments, B is —NHS(O)—. In some embodiments, B is —S(O)NH—. Insome embodiments, B is —C(O)O—. In some embodiments, B is —OC(O)—. Insome embodiments, B is —S—. In some embodiments, B is ═NH—O—. In someembodiments, B is ═NH—NH—. In some embodiments, B is ═NH—N(C₁-C₂₀alkyl)-.

In some embodiments, R² is a fatty acid. In some embodiments, the fattyacid may be of the following Formulae A1, A2 or A3:

where R¹¹ is CO₂H or H;R¹², R¹³ and R¹⁴ are independently of each other H, OH, CO₂H, —CH═CH₂ or—C═CH;Ak is a branched C₆-C₃₀ alkylene;p, q, and r are independently of each other an integer between 6 and 30;or an amide, an ester or a pharmaceutically acceptable salt thereof.In some embodiments, R² is linear or branched C₁-C₃ alkyl-N₃. In someembodiments, R² is cyclooctynyl. In some embodiments, R² is afluorophore. In some embodiments the fluorophore is of the formula

where n is from 1 to 3 and each m is from 1 to 2. In some embodiments, nis 1. In some embodiments, n is 2. In some embodiments, n is 3. In someembodiments, one m is 1 and the other m is 2. In some embodiments, bothm's are 1. In some embodiments, both m's are 2. In some embodiments, R²is a polysaccharide. In some embodiments, R² is —CH(OCH₃)₂. In someembodiments, R² is

In some embodiments, R² is

In some embodiments, R² is,

In some embodiments, R² is

In some embodiments, R² is

In some embodiments, R² is

In some embodiments, Q is H. In some embodiments, Q is —NO₂. In someembodiments, n is 0. In some embodiments, n is from 1 to 6. In someembodiments, n is 1. In some embodiments, n is 2. In some embodiments, nis 3. In some embodiments, n is 4. In some embodiments, n is 5. In someembodiments, n is 6.

In some embodiments, R² is

In some embodiments, n is 0. In some embodiments, n is from 1 to 6. Insome embodiments, n is 1. In some embodiments, n is 2. In someembodiments, n is 3. In some embodiments, n is 4. In some embodiments, nis 5. In some embodiments, n is 6.

In some embodiments, R² is

In some embodiments, n is 0. In some embodiments, n is from 1 to 6. Insome embodiments, n is 1. In some embodiments, n is 2. In someembodiments, n is 3. In some embodiments, n is 4. In some embodiments, nis 5. In some embodiments, n is 6.

In some embodiments, R² is

In some embodiments, R² is

In compounds of formula II, the R2 groups shown below already includesome embodiments incorporating B in formula I above.

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, R¹ is selected from acetyl,

In some embodiments, R¹ is selected from acetyl,

In some embodiments, R¹ is selected from acetyl,

In some embodiments, R¹ is selected from acetyl,

In some embodiments, R¹ is selected from

In some embodiments, R¹ is selected from acetyl and

In some embodiments, R¹ is selected from acetyl and

In some embodiments, R¹ is selected from acetyl and

In some embodiments, R¹ is selected from

In some embodiments, R¹ is selected from acetyl,

In some embodiments, R¹ is selected from

In some embodiments, R¹ is acetyl. In some embodiments, R¹ is

In some embodiments, R¹ is,

In some embodiments, R¹ is

In addition, a multifunctional group A-W—B—R² or NH—W—R² may be presentor absent (i.e. when z is 1 or 0, respectively). In embodiments havingNH—W—R², W is selected from C₁-C₆ linear or branched alkyl or linear orbranched polyethylene glycol having a molecular weight of between about40 and about 80,000 amu. In some embodiments, W is selected from C₁-C₆linear alkyl. In some embodiments, W is selected from C₁-C₆ branchedalkyl. In some embodiments, W is selected from linear polyethyleneglycol having a molecular weight of between about 40 and about 10,000amu. In some embodiments, W is selected from linear polyethylene glycolhaving a molecular weight of between about 40 and about 3,000 amu. Insome embodiments, W is selected from linear polyethylene glycol having amolecular weight of between about 40 and about 80 amu. In someembodiments, W is selected from linear or branched polyethylene glycolhaving a molecular weight of between about 2,000 and about 80,000 amu.In some embodiments, the polyethylene glycol is functionalized with aheteroatom (such as oxygen, nitrogen, or sulfur) capable reacting with areagent to form a bond with another heteroatom, carbon, carbonyhl,sulfonyl, thionyl, and the like.

In embodiments having A-W—B—R², W is selected from C₁-C₆ linear orbranched alkyl or linear or branched polyethylene glycol having amolecular weight of between about 40 and about 80,000 amu. In someembodiments, W is selected from C₁-C₆ linear alkyl. In some embodiments,W is selected from C₁-C₆ branched alkyl. In some embodiments, W isselected from linear polyethylene glycol having a molecular weight ofbetween about 40 and about 10,000 amu. In some embodiments, W isselected from linear polyethylene glycol having a molecular weight ofbetween about 40 and about 3,000 amu. In some embodiments, W is selectedfrom linear polyethylene glycol having a molecular weight of betweenabout 40 and about 80 amu. In some embodiments, W is selected fromlinear or branched polyethylene glycol having a molecular weight ofbetween about 2,000 and about 80,000 amu.

In some embodiments, A is absent. In some embodiments, A is —O—. In someembodiments, A is —NH—. In some embodiments, A is —S—.

In some embodiments, B is absent. In some embodiments, B is —O—. In someembodiments, B is —C(O)—. In some embodiments, B is —NH—. In someembodiments, B is —C(O)NH—. In some embodiments, B is —NHC(O)—. In someembodiments, B is, —NHC(O)O—. In some embodiments, B is —OC(O)NH—. Insome embodiments, B is —OC(O)O—. In some embodiments, B is —C═N(OH)—. Insome embodiments, B is —S(O₂)—. In some embodiments, B is —NHS(O₂)—. Insome embodiments, B is —S(O₂)NH—. In some embodiments, B is —S(O)—. Insome embodiments, B is —NHS(O)—. In some embodiments, B is —S(O)NH—. Insome embodiments, B is —C(O)O—. In some embodiments, B is —OC(O)—. Insome embodiments, B is —S—. In some embodiments, B is ═NH—O—. In someembodiments, B is ═NH—NH—. In some embodiments, B is ═NH—N(C₁-C₂₀alkyl)-.

In some embodiments, R² is selected from C₁-C₃ linear or branchedalkyl-N₃, cyclooctynyl, fluorophore,

and a polysaccharide.

In some embodiments, R² is branched or linear C₁-C₃ alkyl-N₃. In someembodiments, R² is branched C₃ alkyl-N₃. In some embodiments, R² islinear C₁-C₃ alkyl-N₃. In some embodiments, R² is C₁ alkyl-N₃. In someembodiments, R² is C₂ alkyl-N₃. In some embodiments, R² is branched C₃alkyl-N₃.

In some embodiments, R² is cyclooctynyl. The point of attachmentrelative to the alkyne functionality group may be at any position solong as the alkyne can be subsequently reacted or functionalized. Forexample, R² can be connected to W or B at the third position, i.e.

the fourth position, i.e.

or the fifth position, i.e.

In some embodiments, R² is fluorophore. Suitable fluorophores includethose that can re-emit light upon light excitation. Typically, thefluorophore contains several conjugated pi-bonds, such as are present inaromatic groups. Examples include fluorescein, rhodamine, Cy dyes suchas Cy5 and Cy7, Alexa dies such as Alexa 750, Alexa 647, and Alexa 488,coumarins, and the like.

In some embodiments, R² is,

In some embodiments, R² is

In some embodiments, R² is

which corresponds to cytotoxic MMAF connected through a carbonyl to W inNH—W—R².

When the multifunctional group A-W—B—R² or NH—W—R² is absent, then theadjacent amino acid residue whether glutamine or glycine terminates withthe carboxylic acid of the residue (the C-terminus of the peptidebackbone of the modifying compound).

Polysaccharides

In some embodiments, R² is polysaccharide. The polysaccharide may be anyantigenic polysaccharide, particularly a polysaccharide from apathogenic organism. Conjugates of these polysaccharides may be usefulfor immunizing a subject against infection caused by the pathogenicorganism. Exemplary polysaccharides are described below. In particular,the polysaccharide may be a bacterial polysaccharide, e.g. a bacterialcapsular polysaccharide. Representative bacterial polysaccharides aredescribed in Table 1.

TABLE I Polysaccharide Repeat Unit Haemophilus→3)-β-D-Ribf-(I→1)-D-Ribitol-(5→OPO₃→ influenzae Type b (‘PRP’)Neisseria meningitides Group A →6)-α-D-ManpNAc(3OAc)-(I→OPO₃→ Group C→9)-α-D-Neu5Ac(7/8OAc)-(2→ Group W135→6)-α-D-Galp-(I→4)-α-D-Neu5Ac(9OAc)-2→ Group Y→6)-α-D-Glcp-(I→4)-α-D-Neu5Ac(9OAc)-2→ Salmonella→-α-D-GalpNacA(3OAc)-(I→ enterica Typhi Vi Streptococcus pneumoniae Type1 →3)-D-AAT-α-Galp-(I→4)-α-D-GalpA(2/3OAc)- (I→3)-α-D-GalpA-(I→ Type 2→4-β-D-Glcp-(I→3)-[α-D-GlcpA-(I→6)-α-D- Glcp-(I→2)]-α-L-Rhap-(I→3)-α-L-Rhap-(I→3)-β-L-Rhap-(I→ Type 3 →3)-β-D-GlcA-(I→4)-β-D-Glcp-(I→Type 4 →3β-D-ManpNAc-(I→3)-α-L-FucpNAc-(I→3)-α- D-GalpNAc-(I→4)-α-

The polysaccharides may be used in the form of oligosaccharides. Theseare conveniently formed by fragmentation of purified polysaccharide(e.g. by hydrolysis), which will usually be followed by purification ofthe fragments of the desired size.

Polysaccharides may be purified from natural sources. As an alternativeto purification, polysaccharides may be obtained by total or partialsynthesis.

N. meningitidis Capsular Polysaccharides

The polysaccharide may be a bacterial capsular polysaccharide. Exemplarybacterial capsular polysaccharides include those from N. meningitidis.Based on the organism's capsular polysaccharide, various serogroups ofN. meningitidis have been identified, including A, B, C, H, I, K, L,29E, W135, X, Y, and Z. The polysaccharide may be from any of theseserogroups. Typically, the polysaccharide is from one of the followingmeningococcal serogroups: A, C, W135 and Y.

The capsular polysaccharides will generally be used in the form ofoligosaccharides. These are conveniently formed by fragmentation ofpurified capsular polysaccharide (e.g. by hydrolysis), which willusually be followed by purification of the fragments of the desiredsize. Fragmentation of polysaccharides is typically performed to give afinal average degree of polymerization (DP) in the oligosaccharide ofless than 30 (e.g. between 10 and 20, for example around 10 forserogroup A; between 15 and 25 for serogroups W135 and Y, for examplebetween 15 and 20; between 12 and 22 for serogroup C; etc.). DP canconveniently be measured by ion exchange chromatography or bycolorimetric assays (Ravenscroft et al. Vaccine 17, 2802-2816 (1999)).

If hydrolysis is performed, the hydrolysate will generally be sized inorder to remove short-length oligosaccharides (Costantino et al. Vaccine17, 1251-1263 (1999)). This can be achieved in various ways, such asultrafiltration followed by ion-exchange chromatography.Oligosaccharides with a degree of polymerization of less than or equalto about 6 can be removed for serogroup A, and those less than around 4can be removed for serogroups W135 and Y.

Chemical hydrolysis of saccharides generally involves treatment witheither acid or base under conditions that are standard in the art.Conditions for depolymerization of capsular polysaccharides to theirconstituent monosaccharides are known in the art. One depolymerizationmethod involves the use of hydrogen peroxide (see WO02/058737 which isincorporated herein by reference).

Hydrogen peroxide is added to a saccharide (e.g. to give a final H₂O₂concentration of 1%), and the mixture is then incubated (e.g. at around55° C.) until a desired chain length reduction has been achieved. Thereduction over time can be followed by removing samples from the mixtureand then measuring the (average) molecular size of saccharide in thesample. Depolymerization can then be stopped by rapid cooling once adesired chain length has been reached

Serogroups C, W135 and Y

Techniques for preparing capsular polysaccharides from meningococci havebeen known for many years, and typically involve a process comprisingthe steps of polysaccharide precipitation (e.g. using a cationicdetergent), ethanol fractionation, cold phenol extraction (to removeprotein) and ultracentrifugation (to remove LPS) (for example, seeFrash, Advances in Biotechnological Processes 13, 123-145 (1990) (eds.Mizrahi & Van Wezel).

One such process involves polysaccharide precipitation followed bysolubilization of the precipitated polysaccharide using a lower alcohol(see WO03/007985 which is incorporated herein by reference).

Precipitation can be achieved using a cationic detergent such astetrabutylammonium and cetyltrimethylammonium salts (e.g. the bromidesalts), or hexadimethrine bromide and myristyltrimethylammonium salts.Cetyltrimethylammonium bromide (‘CTAB’) is particularly preferred(Inzana, Infect. Immun. 55, 1573-1579 (1987). Solubilization of theprecipitated material can be achieved using a lower alcohol such asmethanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol,2-methyl-propan-1-ol, 2-methyl-propan-2-ol, diols, etc., but ethanol isparticularly suitable for solubilizing CTAB-polysaccharide complexes.Ethanol may be added to the precipitated polysaccharide to give a finalethanol concentration (based on total content of ethanol and water) ofbetween 50% and 95%.

After re-solubilization, the polysaccharide may be further treated toremove contaminants. This is particularly important in situations whereeven minor contamination is not acceptable (e.g. for human vaccineproduction). This will typically involve one or more steps of filtratione.g. depth filtration, filtration through activated carbon may be used,size filtration and/or ultrafiltration. Once filtered to removecontaminants, the polysaccharide may be precipitated for furthertreatment and/or processing. This can be conveniently achieved byexchanging cations (e.g. by the addition of calcium or sodium salts).

As an alternative to purification, capsular polysaccharides of thepresent invention may be obtained by total or partial synthesis e.g. Hibsynthesis is disclosed in Kandil et al. Glvcoconi J 14, 13-17. (1997),and MenA synthesis in Berkin et al. Chemistry 8, 4424-4433 (2002).

The polysaccharide may be chemically modified, that is it may be0-acetylated or de-O-acetylated. Any such de-O-acetylation orhyper-acetylation may be at specific positions in the polysaccharide.For instance, most serogroup C strains have O-acetyl groups at positionC-7 and/or C-8 of the sialic acid residues, but about 15% of clinicalisolates lack these O-acetyl groups (Glode et al. J Infect Pis 139,52-56 (1979); see also WO94/05325 and U.S. Pat. No. 5,425,946 that areincorporated herein by reference). The acetylation does not seem toaffect protective efficacy (e.g. unlike the Menjugate™ product, theNeisVac-C™ product uses a de-O-acetylated polysaccharide, but bothvaccines are effective). The serogroup W135 polysaccharide is a polymerof sialic acid-galactose disaccharide units. The serogroup Ypolysaccharide is similar to the serogroup W135 polysaccharide, exceptthat the disaccharide repeating unit includes glucose instead ofgalactose. Like the serogroup C polysaccharides, the MenW135 and MenYpolysaccharides have variable 0-acetylation, but at sialic acid 7 and 9positions (see WO2005/033148 which is incorporated herein by reference).Any such chemical modifications preferably take place beforeconjugation, but may alternatively or additionally take place duringconjugation.

Polysaccharides from different serogroups can be purified separately,and may then be combined either before or after conjugation.

Serogroup A

The polysaccharide may be from a serogroup A. The polysaccharide can bepurified in the same way as for serogroups C, W135 and Y (see above),although it is structurally different, whereas the capsules ofserogroups C, W135 and Y are based around sialic acid(N-acetyl-neuraminic acid, NeuAc), the capsule of serogroup A is basedon N-acetyl-mannosamine, which is the natural precursor of sialic acid.The serogroup A polysaccharide is particularly susceptible tohydrolysis, and its instability in aqueous media means that (a) theimmunogenicity of liquid vaccines against serogroup A declines overtime, and (b) quality control is more difficult, due to release ofsaccharide hydrolysis products into the vaccine.

Native MenA capsular polysaccharide is a homopolymer of (a1→6)-linkedN-acetyl-D-mannosamine-1-phosphate, with partial O-acetylation at C3 andC4. The principal glycosidic bond is a 1-6 phosphodiester bond involvingthe hemiacetal group of C1 and the alcohol group of C6 of theD-mannosamine. The average chain length is 93 monomers. It has thefollowing formula:

A modified polysaccharide has been prepared which retains theimmunogenic activity of the native serogroup A polysaccharide but whichis much more stable in water. Hydroxyl groups attached at carbons 3 and4 of the monosaccharide units are replaced by a blocking group (seeWO03/080678 & WO2008/084411).

The number of monosaccharide units having blocking groups in place ofhydroxyls can vary. For example, all or substantially all themonosaccharide units may have blocking groups. Alternatively, at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the monosaccharideunits may have blocking groups. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 monosaccharide units may have blocking groups.

Likewise, the number of blocking groups on a monosaccharide unit mayvary. For example, the number of blocking groups on any particularmonosaccharide unit may be 1 or 2.

The terminal monosaccharide unit may or may not have a blocking groupinstead of its native hydroxyl. It is preferred to retain a freeanomeric hydroxyl group on a terminal monosaccharide unit in order toprovide a handle for further reactions (e.g. conjugation). Anomerichydroxyl groups can be converted to amino groups (—NH₂ or —NH-E, where Eis a nitrogen protecting group) by reductive amination (using, forexample, NaBH₃CN/NH₄CI), and can then be regenerated after otherhydroxyl groups have been converted to blocking groups.

Blocking groups to replace hydroxyl groups may be directly accessiblevia a derivatizing reaction of the hydroxyl group i.e. by replacing thehydrogen atom of the hydroxyl group with another group. Suitablederivatives of hydroxyl groups which act as blocking groups are, forexample, carbamates, sulfonates, carbonates, esters, ethers (e.g. silylethers or alkyl ethers) and acetals. Some specific examples of suchblocking groups are allyl, Aloe, benzyl, BOM, t-butyl, trityl, TBS,TBDPS, TES, TMS, TIPS, PMB, MEM, MOM, MTM, THP, etc. Other blockinggroups that are not directly accessible and which completely replace thehydroxyl group include C₁₋₁₂ alkyl, C₃₋₁₂ alkyl, C₅₋₁₂ aryl, C₅₋₁₂ arylC₁₋₆ alkyl, NR²¹R²² (R²¹ and R²² are defined in the followingparagraph), H, F, C, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃, CCI₃, etc.

Typical blocking groups are of the formula: —O—X′—Y′ and —OR²³ wherein:X′ is C(O), S(O) or SO₂; Y is C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₃₋₁₂cycloalkyl, C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each of which mayoptionally be substituted with 1, 2 or 3 groups independently selectedfrom F, C, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCI₃; or Y′ is NR²¹R²²;R²¹ and R²² are independently selected from H, C₁₋₁₂ alkyl, C₃₋₁₂cycloalkyl, C₅₋₁₂ aryl, C₅₋₁₂ aryl-C₁₋₆ alkyl; or R²¹ and R²² may bejoined to form a C₃₋₁₂ saturated heterocyclic group; R²³ is C₁₋₁₂ alkylor C₃₋₁₂ cycloalkyl, each of which may optionally be substituted with 1,2 or 3 groups independently selected from F, Cl, Br, CO₂(C₁₋₆ alkyl),CN, CF₃ or CCl₃; or R²³ is C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each ofwhich may optionally be substituted with 1, 2, 3, 4 or 5 groups selectedfrom F, Cl, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCI₃. When R²³ isC₁₋₁₂ alkyl or C₃₋₁₂ cycloalkyl, it is typically substituted with 1, 2,or 3 groups as defined above. When R²¹ and R²² are joined to form aC₃₋₁₂ saturated heterocyclic group, it is meant that R²¹ and R²²together with the nitrogen atom form a saturated heterocyclic groupcontaining any number of carbon atoms between 3 and 12 (e.g. C₃, C₄, C₅,C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂). The heterocyclic group may contain 1 or2 heteroatoms (such as N, O, or S) other than the nitrogen atom.Examples of C₃₋₁₂ saturated heterocyclic groups are pyrrolidinyl,piperidinyl, morpholinyl, piperazinyl, imidazolidinyl, azetidinyl andaziridinyl.

Blocking groups —O—X—Y and —OR²³ can be prepared from —OH groups bystandard derivatizing procedures, such as reaction of the hydroxyl groupwith an acyl halide, alkyl halide, sulfonyl halide, etc. Hence, theoxygen atom in —O—X′—Y′ is usually the oxygen atom of the hydroxylgroup, while the —X′—Y′ group in —O—X′—Y′ usually replaces the hydrogenatom of the hydroxyl group.

Alternatively, the blocking groups may be accessible via a substitutionreaction, such as a Mitsonobu-type substitution. These and other methodsof preparing blocking groups from hydroxyl groups are well known.

Specific blocking groups for use in the invention are —OC(O)CF₃ (Nilsson& Svensson Carbohydrate Research 69, 292-296 (1979)) and a carbamategroup OC(O)NR²¹R²², where R²¹ and R²² are independently selected fromC₁₋₆ alkyl. Typically, R²¹ and R²² are both methyl i.e. the blockinggroup is —OC(O)NMe₂. Carbamate blocking groups have a stabilizing effecton the glycosidic bond and may be prepared under mild conditions.

A particularly preferred blocking group is —OC(O)CH₃ (seeWO2008/084411). The proportion of 4- and/or 3-positions in the modifiedNeisseria meningitidis serogroup A polysaccharide that have thisblocking group may vary. For example, the proportion of 4-positions thathave blocking groups may be about 0%, at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or about 100%, with at least 80% and about 100%being preferred. Similarly, the proportion of 3-positions that haveblocking groups may be about 0%, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or about 100%, with at least 80% and about 100% beingpreferred. Typically, the proportion of 4- and 3-positions that haveblocking groups is about the same at each position. In other words, theratio of 4-positions that have blocking groups to 3-positions that haveblocking groups is about 1:1. However, in some embodiments, theproportion of 4-positions that have blocking groups may vary relative tothe proportion of 3-positions that have blocking groups. For example,the ratio of 4-positions that have blocking groups to 3-positions thathave blocking groups may be 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14,1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or 1:2.Similarly, the ratio of 3-positions that have blocking groups to4-positions that have blocking groups may be 1:20, 1:19, 1:18, 1:17,1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,1:3 or 1:2.

Typical modified MenA polysaccharides contain n monosaccharide units,where at least h % of the monosaccharide units do not have —OH groups atboth of positions 3 and 4. The value of h is 24 or more (e.g. 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98,99 or 100) and is usually 50 or more. The absent —OH groups are blockinggroups as defined above.

Other typical modified MenA polysaccharides comprise monosaccharideunits, wherein at least s of the monosaccharide units do not have —OH atthe 3 position and do not have —OH at the 4 position. The value of s isat least 1 (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90). The absent —OH groups are blocking groups as defined above.

Suitable modified MenA polysaccharides have the formula:

wherein p is an integer from 1 to 100 (particularly an integer from 5 to25, usually 15-25); T is of the formula (A) or (B):

each Z group is independently selected from OH or a blocking group asdefined above; and each Q group is independently selected from OH or ablocking group as defined above;

Y is selected from OH or a blocking group as defined above;

E is H or a nitrogen protecting group; and wherein more than about 7%(e.g. 8%, 9%, 10% or more) of the Q groups are blocking groups. In someembodiments, the hydroxyl group attached at carbon 1 in formula (A) isreplaced by a blocking group as defined above. In some embodiments, E informula (B) is a linker or a carrier molecule as discussed below. When Eis a linker, the linker may be covalently bonded to a carrier molecule.

Each of the p+2 Z groups may be the same or different from each other.Likewise, each of the n+2 Q groups may be the same or different fromeach other. All the Z groups may be OH. Alternatively, at least 10%, 20,30%, 40%, 50% or 60% of the Z groups may be OAc. Typically, about 70% ofthe Z groups are OAc, with the remainder of the Z groups being OH orblocking groups as defined above. At least about 7% of Q groups areblocking groups. Typically, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or even 100% of the Q groups are blocking groups.

Glucans

The polysaccharide may be a glucan. Glucans are glucose-containingpolysaccharides found inter alia in fungal cell walls. The oglucansinclude one or more a-linkages between glucose subunits, whereasβ-glucans include one or more β-linkages between glucose subunits. Theglucan used in accordance with the invention includes β linkages, andmay contain only β linkages (i.e. no a linkages).

The glucan may comprise one or more β-1,3-linkages and/or one or moreβ-1,6-linkages. It may also comprise one or more β-1,2-linkages and/or(β-1,4-linkages, but normally its only β linkages will be β-1,3-linkagesand/or β-1 6-linkages. The glucan may be branched or linear. Full-lengthnative β-glucans are insoluble and have a weight average molecularweight in the megadalton range. Thus, it is better to use solubleglucans in conjugates. Solubilization may be achieved by fragmentinglong insoluble glucans. This may be achieved by hydrolysis or, moreconveniently, by digestion with a glucanase (e.g. with a β-1,3-glucanaseor a (β-1,6-glucanase). As an alternative, short glucans can be preparedsynthetically by joining monosaccharide building blocks.

Low molecular weight glucans are preferred, particularly those with aweight average molecular weight of less than 100 kDa (e.g. less than 80,70, 60, 50, 40, 30, 25, 20, or 15 kDa). It is also possible to useoligosaccharides e.g. containing 60 or fewer (e.g. 59, 58, 57, 56, 55,54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 39, 38, 37,36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4) glucosemonosaccharide units. Within this range, oligosaccharides with between10 and 50 or between 20 and 40 monosaccharide units. The glucan may be afungal glucan. A fungal glucan will generally be obtained from a fungusbut, where a particular glucan structure is found in both fungi andnon-fungi (e.g. in bacteria, lower plants or algae) then the non-fungalorganism may be used as an alternative source. Thus the glucan may bederived from the cell wall of a Candida, such as C. albicans, or fromCoccidioides immitis, Trichophyton verrucosum, Blastomyces dermatidis,Cryptococcus neoformans, Histoplasma capsulatum, Saccharomycescerevisiae, Paracoccidioides brasiliensis, or Pythiumn insidiosum.

There are various sources of fungal β-glucans. For instance, pureβ-glucans are commercially available e.g. pustulan (Calbiochem) is aβ-1,6-glucan purified from Umbilicaria papullosa. β-glucans can bepurified from fungal cell walls in various ways. Tokunaka et al.Carbohydrate Research 316, 161-172. (1999), for instance, discloses atwo-step procedure for preparing a water-soluble β-glucan extract fromCandida, free from cell-wall mannan, involving NaCIO oxidation and DMSOextraction. The resulting product (′Candida soluble β-D-glucan′ or‘CSBG’) is mainly composed of a linear β-1,3-glucan with a linearβ-1,6-glucan moiety. Similarly, WO03/097091 discloses the production ofGG-zym from Calbicans. Such glucans from C. albicans, include (a)β-1,6-glucans with β-1,3-glucan lateral chains and an average degree ofpolymerization of about 30, and (b) β-1,3-glucans with (β-1,6-glucanlateral chains and an average degree of polymerization of about 4.

In some embodiments, the glucan is a β-1,3 glucan with some β-1,6branching, as seen in e.g. laminarins. Laminarins are found in brownalgae and seaweeds. The β(1-3):β(1-6) ratios of laminarins vary betweendifferent sources e.g. it is as low as 3:2 in Eisenia bicyclislaminarin, but as high as 7:1 in Laminaria digititata laminarin (Pang etal. Biosci Biotechnol Biochem 69, 553-8 (2005)). Thus the glucan mayhave a β(1-3):β(1-6) ratio of between 1.5:1 and 7.5:1 e.g. about 2:1,3:1, 4:1, 5:1, 6:1 or 7:1. Optionally, the glucan may have a terminalmannitol subunit, e.g. a 1,1-O-linked mannitol residue (Read et al.Carbohydr Res. 281, 187-201 (1996). The glucan may also comprise mannosesubunits.

In other embodiments, the glucan has exclusively or mainly β-1,3linkages, as seen in curdlan. These glucans may elicit better protectionthan glucans comprising other linkages, particularly glucans comprisingβ-1,3 linkages and a greater proportion of β-1,6 linkages. Thus theglucan may be made solely of β-1,3-linked glucose residues (e.g. linearβ-D-glucopyranoses with exclusively 1,3 linkages). Optionally, though,the glucan may include monosaccharide residues that are not β-1,3-linkedglucose residues e.g. it may include β-1,6-linked glucose residues. Theratio of β-1,3-linked glucose residues to these other residues should beat least 8:1(e.g. >9:1, >10:1, >11:1, >12:1, >13:1, >14:1, >15:1, >16:1, >17:1, >18:1, >19:1, >20:1, >25:1, >30:1, >35:1, >40:1, >45:1, >50:1, >75:1, >100:1,etc.) and/or there are one or more(e.g. >1, >2, >3, >4, >5, >6, >7, >8, >9, >10, >11, >12, etc.) sequencesof at least five(e.g. >5, >6, >7, >8, >9, >10, >11, >12, >13, >14, >15, >16, >17, >18, >19, >20, >30, >40, >50, >60,etc.) adjacent non-terminal residues linked to other residues only byβ-1,3 linkages. By “non-terminal” it is meant that the residue is notpresent at a free end of the glucan. In some embodiments, the adjacentnon-terminal residues may not include any residues coupled to a carriermolecule or linker. The presence of five adjacent non-terminal residueslinked to other residues only by β-1,3 linkages may provide a protectiveantibody response, e.g. against C. albicans.

In further embodiments, a conjugate may include two different glucanse.g. a first glucan having a β(1-3):β(1-6) ratio of between 1.5:1 and7.5:1, and a second glucan having exclusively or mainly β-1,3 linkages.For instance a conjugate may include both a laminarin glucan and acurdlan glucan. Where a β-glucan includes both β-1,3 and β-1,6 linkagesat a desired ratio and/or sequence then this glucan may be found innature (e.g. a laminarin), or it may be made artificially. For instance,it may be made by chemical synthesis, in whole or in part.

Methods for the chemical synthesis of β-1,3/3-1,6 glucans are known, forexample from Takeo and Tei Carbohvdr Res. 145, 293-306 (1986), Tanaka etal. Tetrahedron Letters 44, 3053-3057 (2003), Ning et al. TetrahedronLetters 43, 5545-5549 (2002), Geurtsen et al. Journal of OrganicChemistry 64 (21):7828-7835 (1999), Wu et al. Carbohvdr Res. 338,2203-12 (2003), Nicolaou et al. J. Am. Chem. Soc. 119, 449-450 (1997),Yamada et al. Tetrahedron Letters 40, 4581-4584 (1999), Yamago et al.Org. Lett. 24, 3867-3870 (2001), Yuguo et al. Tetrahedron 60, 6345-6351(2004), Amaya et al. Tetrahedron Letters 42:9191-9194 (2001), Mei et al.Carbohvdr Res. 340. 2345-2351 (2005).

β-glucan including both β-1,3 and β-1,6 linkages at a desired ratio mayalso be made starting from an available glucan and treating it with aβ-1,6-glucanase (also known as glucan endo-1,6-β-glucosidase,1,6-β-D-glucan glucanohydrolase, etc.; EC 3.2.1.75) or a β-1,3-glucanase(such as an exo-1,3-glucanase (EC 3.2.1.58) or an endo-1,3-glucanase (EC3.2.1.39) until a desired ratio and/or sequence is reached.

When a glucan containing solely β-1,3-linked glucose is desired then3-1,6-glucanase treatment may be pursued to completion, asβ-1,6-glucanase will eventually yield pure β-1,3 glucan. Moreconveniently, however, a pure β-1,3-glucan may be used. These may bemade synthetically, by chemical and/or enzymatic synthesis e.g. using a(1→3)-β-D-glucan synthase, of which several are known from manyorganisms (including bacteria, yeasts, plants and fungi). Methods forthe chemical synthesis of β-1,3 glucans are known, for example fromTakeo et al. Carbohydr Res. 245, 81-96 (1993), Jamois et al.Glycobiology 15(4), 393-407 (2005), Lefeber et al. C em. Eur. J.7(20):441 1-4421 (2001) and Huang et al. Carbohydr Res. 340, 603-608(2005). As a useful alternative to synthesis, a natural β-1,3-glucan maybe used, such as a curdlan (linear (β-1,3-glucan from an Agrobacteriumpreviously known as Alcaligenes faecalis var. myxogenes; commerciallyavailable e.g. from Sigma-Aldrich catalog C7821) or paramylon(β-1,3-glucan from Euglena). Organisms producing high levels ofβ-1,3-glucans are known in the art e.g. the Agrobacterium of U.S. Pat.No. 5,508,191 or MiKyoung et al. Biochemical Engineering Journal. 16,163-8 (2003), or the Euglena gracilis of Barsanti et al. J App.Phycology, 13, 59-65 (2001).

Laminarin and curdlan are typically found in nature as high molecularweight polymers e.g. with a weight average molecular weight of at least100 kDa. They are often insoluble in aqueous media. In their naturalforms, therefore, they are not well suited to immunization. Thus, insome embodiments, a shorter glucan e.g. those containing 60 or fewerglucose monosaccharide units (e.g. 59, 58, 57, 56, 55, 54, 53, 52, 51,50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4). A glucan having a number ofglucose residues in the range of 2-60 may be used e.g. between about10-50 or between about 20-40 glucose units. A glucan with 25-30 glucoseresidues is particularly useful. Suitable glucans may be formed e.g. byacid hydrolysis of a natural glucan, or by enzymatic digestion e.g. witha glucanase, such as a β-1,3-glucanase. A glucan with 11-19, e.g. 13-19and particularly 15 or 17, glucose monosaccharide units is also useful.In particular, glucans with the following structures (A) or (B) arespecifically envisaged for use: (A)

wherein s+2 is in the range of 2-60, e.g. between 10-50 or between 2-40.

In some embodiments, s+2 is in the range of 25-30 or 1 1-19, e.g. 13-17.In particular, s+2=15 is suitable. In addition, s+2=6 is suitable.

wherein t is in the range of 0-9, e.g. between 1-7 or between 2-6.Preferably, t is in the range of 3-4 or 1-3. In particular, t=2 issuitable. The * and ** indicate the respective attachment points of thepolysaccharide units.

In some embodiments, the glucan contains between 5 to 7 glucosemonosaccharide units (i.e. 5, 6 or 7). In particular, a glucan having 6glucose monosaccharide units may be preferred. For example, the glucanmay be a curdlan having 6 glucose monosaccharide units.

In some embodiments, the glucan is a single molecular species. In theseembodiments, all of the glucan molecules are identical in terms ofsequence.

Accordingly, all of the glucan molecules are identical in terms of theirstructural properties, including molecular weight etc. Typically, thisform of glucan is obtained by chemical synthesis, e.g. using the methodsdescribed above. Alternatively, in other embodiments, the glucan may beobtained from a natural glucan, e.g. a glucan from L. digitata,Agrobacterium or Euglena as described above, with the glucan beingpurified until the required single molecular species is obtained.Natural glucans that have been purified in this way are commerciallyavailable. A glucan that is a single molecular species may be identifiedby measuring the polydispersity (Mw/Mn) of the glucan sample. Thisparameter can conveniently be measured by SEC-MALLS, for example asdescribed in Bardotti et al. Vaccine 26, 2284-96 (2008). Suitableglucans for use in this embodiment of the invention have apolydispersity of about 1, e.g. 1.01 or less.

Solubility of natural glucans, such as curdlan, can be increased byintroducing ionic groups (e.g. by sulfation, particularly at 0-6 incurdlan). Such modifications may be used with the invention, but areideally avoided as they may alter the glucan's antigenicity.

When the polysaccharide is a glucan, it is typically a laminarin.

S. pneumoniae Capsular Polysaccharides

As discussed above, the polysaccharide may also be a bacterial capsularpolysaccharide. Further exemplary bacterial capsular polysaccharidesinclude those from S. pneumoniae. When the polysaccharide is a capsularpolysaccharide from S. pneumoniae, it is typically from one of thefollowing pneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V,10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Insome embodiments, it is from 1, 5, 6B, 14, 19F, and 23F. Capsularpolysaccharides from S. pneumoniae comprise repeating oligosaccharideunits which may contain up to 8 sugar residues. The oligosaccharideunits for the main S. pneumoniae serotypes are described in the tableabove, Jones An. Acad. Bras. Cienc, 77(2), 293-324 (2005) and Jones, JPharm Biomed Anal 38, 840-850 (2005).

S. agalactiae Capsular Polysaccharides

Further exemplary bacterial capsular polysaccharides include those fromStreptococcus agalactiae (“GBS”). The capsular polysaccharide iscovalently linked to the peptidoglycan backbone of GBS, and is distinctfrom the group B antigen, which is another polysaccharide that isattached to the peptidoglycan backbone.

The GBS capsular polysaccharides are chemically related, but areantigenically very different. All GBS capsular polysaccharides share thefollowing trisaccharide core: β-D-GlcpNAc(1→3)β-D-Galp(1→4)β-D-Glcp

The various GBS serotypes differ by the way in which this core ismodified. The difference between serotypes 1a and III, for instance,arises from the use of either the GlcNAc (1a) or the Gal (III) in thiscore for linking consecutive trisaccharide cores.

Serotypes 1a and 1b both have a [a-D-NeupNAc(2→3)β-D-Galp-(1→]disaccharide linked to the GlcNAc in the core, but the linkage is either1→4 (1a) or 1→3 (1b).

GBS-related disease arises primarily from serotypes Ia, Ib, II, III, IV,V, VI, VII, and VIII, with over 85% being caused by five serotypes: Ia,Ib, III & V. A polysaccharide from one of these four serotypes may beused. The capsular polysaccharides of each of these four serotypesinclude: (a) a terminal N-acetyl-neuraminic acid (NeuNAc) residue(commonly referred to as sialic acid), which in all cases is linked 2→3to a galactose residue; and (b) a N-acetyl-glucosamine residue (GlcNAc)within the trisaccharide core.

All four polysaccharides include galactose residues within thetrisaccharide core, but serotypes Ia, Ib, II & III also containadditional galactose residues in each repeating unit.

Polysaccharides used may be in their native form, or may have beenmodified. For example, the polysaccharide may be shorter than the nativecapsular polysaccharide, or may be chemically modified. In particular,the serotype V capsular polysaccharide used in the invention may bemodified as described in WO2006/050341 and Guttormsen et al. Proc NatlAcad Sci USA. 105(15), 5903-8 (2008) Epub 2008 Mar. 31. For example, aserotype V capsular polysaccharide that has been substantiallydesialylated. Desialylated GBS serotype V capsular polysaccharide may beprepared by treating purified GBS serotype V capsular polysaccharideunder mildly acidic conditions (e.g. 0.1 M sulphuric acid at 80° C. for60 minutes) or by treatment with neuraminidase. Thus the polysaccharideused according to the invention may be a substantially full-lengthcapsular polysaccharide, as found in nature, or it may be shorter thanthe natural length. Full-length polysaccharides may be depolymerized togive shorter fragments for use with the invention e.g. by hydrolysis inmild acid, by heating, by sizing chromatography, etc. In particular, theserotype II and/or III capsular polysaccharides used in the inventionmay be depolymerized as described in WO96/40795 and Michon et al. ClinVaccine Immunol. (2006) 13(8), 936-43.

The polysaccharide may be chemically modified relative to the capsularpolysaccharide as found in nature. For example, the polysaccharide maybe de-O-acetylated (partially or fully), de-N-acetylated (partially orfully), N-propionated (partially or fully), etc. De-acetylation mayoccur before, during or after conjugation, but preferably occurs beforeconjugation. Depending on the particular polysaccharide, de-acetylationmay or may not affect immunogenicity. The relevance of O-acetylation onGBS polysaccharides in various serotypes is discussed in Lewis et al.PNAS USA 101, 11123-8 (2004), and in some embodiments O-acetylation ofsialic acid residues at positions 7, 8 and/or 9 is retained before,during and after conjugation e.g. by protection/de-protection, byre-acetylation, etc. However, typically the GBS polysaccharide used inthe present invention has substantially no O-acetylation of sialic acidresidues at positions 7, 8 and/or 9. In particular, when the GBSpolysaccharide has been purified by base extraction as described below,then 0-acetylation is typically lost. The effect of de-acetylation etc.can be assessed by routine assays.

Capsular polysaccharides can be purified by known techniques, asdescribed in Wessels et al. Infect Immun 57, 1089-94 (1989). A typicalprocess involves base extraction, centrifugation, filtration,RNase/DNase treatment, protease treatment, concentration, size exclusionchromatography, ultrafiltration, anion exchange chromatography, andfurther ultrafiltration. Treatment of GBS cells with the enzymemutanolysin, which cleaves the bacterial cell wall to free the cell wallcomponents, is also useful.

As an alternative, the purification process described in WO2006/082527can be used. This involves base extraction, ethanol/CaCl2 treatment,CTAB precipitation, and re-solubilisation. A further alternative processis described in WO2009/081276.

S. aureus Capsular Polysaccharides

Further exemplary bacterial capsular polysaccharides include those fromS. aureus, particularly the capsular polysaccharides of S. aureus type 5and type 8. The structures of type 5 and type 8 capsular polysaccharideswere described in Moreau et al. Carbohydrate Res. 339(5), 285-91 (1990)and Fournier et al. Infect. Immun. 45(1), 87-93 (1984) as:

Type 5

→4)-β-D-ManNAcA(30Ac)-(1→4)-a-L-FucNAc(1→3)-β-D-FucNAc-(1

Type 8

→3)-β-D-ManNAcA(40Ac)-(1→3)-a-L-FucNAc(1→3)-β-D-FucNAc-(1 Recent NMRspectroscopy data (Jones Carbohydrate Res. 340(6), 1097-106 (2005)) hasled to a revision of these structures to:

Type 5

→4)-β-D-ManNAcA-(1→4)-a-L-FucNAc(30Ac)-(1→3)-β-D-FucNAc-(1

Type 8

→3)-β-D-ManNAcA(40Ac)-(1→3)-a-L-FucNAc(1→3)-a-D-FucNAc(1→Thepolysaccharide may be chemically modified relative to the capsularpolysaccharide as found in nature.

For example, the polysaccharide may be de-O-acetylated (partially orfully), de-N-acetylated (partially or fully), N-propionated (partiallyor fully), etc. De-acetylation may occur before, during or afterconjugation, but typically occurs before conjugation. The effect ofde-acetylation etc. can be assessed by routine assays. For example, therelevance of O-acetylation on S. aureus type 5 or type 8 capsularpolysaccharides is discussed in Fattom et al. Infect Immun.66(10):4588-92 (1998). The native polysaccharides are said in thisdocument to have 75% O-acetylation. These polysaccharides inducedantibodies to both the polysaccharide backbone and O-acetyl groups.Polysaccharides with 0% O-acetylation still elicited antibodies to thepolysaccharide backbone. Both types of antibody were opsonic against S.aureus strains that varied in their O-acetyl content. Accordingly, thetype 5 or type 8 capsular polysaccharides used in the present inventionmay have between 0 and 100% 0-acetylation.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (e.g. asdescribed in Lemercinier and Jones Carbohydrate Res. 296, 83-96 (1996),Jones and Lemercinier, J Pharm BiomedAnal. 30(4), 1233-47 (2002),WO05/033148 or WO 00/56357. A further method is described in Hestrin J.Biol. Chem. 180, 249-261 (1949). Similar methods may be used todetermine the degree of N-acetylation of the polysaccharide. O-acetylgroups may be removed by hydrolysis, for example by treatment with abase such as anhydrous hydrazine (Konadu et al. Infect. Immun. 62,5048-5054 (1994)) or NaOH (Fattom et al. Infect Immun. 66(10):4588-92(1998)). Similar methods may be used to remove N-acetyl groups. Tomaintain high levels of O-acetylation on type 5 and/or 8 capsularpolysaccharides, treatments that lead to hydrolysis of the 0-acetylgroups are minimized, e.g. treatments at extremes of pH.

Capsular polysaccharides can be purified by known techniques, asdescribed in the references herein. A typical process involvesphenol-ethanol inactivation of S. aureus cells, centrifugation,lysostaphin treatment, RNase/DNase treatment, centrifugation, dialysis,protease treatment, further dialysis, filtration, precipitation withethanol/CaCl2, dialysis, freeze-drying, anion exchange chromatography,dialysis, freeze-drying, size exclusion chromatography, dialysis andfreeze-drying (Fattom et al. Infect Immun. 58(7), 2367-74 (1990)). Analternative process involves autoclaving S. aureus cells,ultrafiltration of the polysaccharide-containing supernatant,concentration, lyophilisation, treatment with sodium metaperiodate toremove teichoic acid, further ultrafiltration, diafiltration, highperformance size exclusion liquid chromatography, dialysis andfreeze-drying (Gilbert et al. J. Microb. Meth. 20, 39-46 (1994)). Theinvention is not limited to polysaccharides purified from naturalsources, however, and the polysaccharides may be obtained by othermethods, such as total or partial synthesis.

Other Bacterial Capsular Polysaccharides

Further exemplary bacterial capsular polysaccharides include those fromHaemophilus influenzae Type b, Salmonella enterica Typhi Vi andClostridium difficile.

S. agalactiae carbohydrate: Non-capsular bacterial polysaccharides mayalso be used. An exemplary non-capsular bacterial polysaccharides is theS. pyogenes GAS carbohydrate (also known as the GAS cell wallpolysaccharide, or GASP). This polysaccharide features a branchedstructure with an L-rhamnopyranose (Rhap) backbone consisting ofalternating alpha-(1→2) and alpha-(1→3) links and D-N-acetylglucosamine(GlcpNAc) residues beta-(1→3)-connected to alternating rhamnose rings(Kreis et al. Int J Biol Macromol. 17(3-4), 117-30 (1995)).

The GAS carbohydrate will generally be in its native form, but it mayhave been modified. For example, the polysaccharide may be shorter thanthe native GAS carbohydrate, or may be chemically modified.

Thus the polysaccharide used according to the invention may be asubstantially full-length GAS carbohydrate, as found in nature, or itmay be shorter than the natural length. Full-length polysaccharides maybe depolymerized to give shorter fragments for use with the inventione.g. by hydrolysis in mild acid, by heating, by sizing chromatography,etc. A short fragment thought to correspond to the terminal unit on theGAS carbohydrate has been proposed for use in a vaccine (Hoog et al.,Carbohydr Res. 337(21-23), 2023-36 (2002)). Accordingly, short fragmentsare envisaged in the present invention. However, it is preferred to usepolysaccharides of substantially full-length. The GAS carbohydratetypically has a weight average molecular weight of about 10 kDa, inparticular about 7.5-8.5 kDa. Molecular masses can be measured by HPLC,for example SEC-HPLC using a TSK Gel G3000SW column (Sigma) relative topullulan standards, such as those available from Polymer StandardService (www.Polymer.de).

The polysaccharide may be chemically modified relative to the GAScarbohydrate as found in nature. For example, the polysaccharide may bede-N-acetylated (partially or fully), N-propionated (partially orfully), etc. The effect of de-acetylation etc., for example onimmunogenicity, can be assessed by routine assays.

In some embodiments, the polysaccharide is GBSII antigenticpolysaccharide having the structure shown below:

In some embodiments, the polysaccharide is GBSV antigenticpolysaccharide having the structure shown below:

In some embodiments, the polysaccharide is MenA antigenic polysaccharidehaving the structure shown below:

In another aspect, compounds of the formula (I)R¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) or (II)R¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z) are disclosed where x, y, z,R¹, R², A, B, and W are defined above. In some embodiments, thecompounds are any one of:

In embodiments having an eight-membered cycloalkyne group, that groupcan be attached to the modifying group by covalent linkage. Typically,the eight-membered cycloalkyne group is attached via a spacer and at aterminus of the spacer. The other terminus of the spacer has afunctional group for attachment to the modifying group through the aminoor carboxylic acid terminus of the peptide, but not at the ε-amino groupof the glutamine. For example, if attachment will be at the amineportion of the modifying group, then spacer can include any functionalgroup that allows attachment to an amine (e.g. a succinimidyl ester).Similarly, if the attachment will be at the carboxylic acid portion ofthe modifying group, then the spacer can include any functional groupthat allows attachment to a carboxylic acid (e.g. an amine).

In some embodiments, the eight-membered cycloalkyne group includes oneor more nitrogen atoms, such as 1, 2 or 3 nitrogen atoms. In someembodiments, the eight-membered cycloalkyne group is fused to one ormore other ring systems, such as cyclopropane or benzene. In onepreferred embodiment, the eight-membered cycloalkyne group is fused to acyclopropane group. In another preferred embodiment, the eight-memberedcycloalkyne group is fused to two benzene groups. In most preferredembodiments, the eight-membered cycloalkyne group is a cyclooctynegroup.

In one embodiment, the attachment is carried out using a compound havingthe formula X¹-L-X², where X¹ is the eight-membered cycloalkyne groupand X²-L is the spacer. In these embodiments, X² may be any group thatcan react with a functional group on the amine group on the peptide, andL is a linking moiety in the spacer.

In one embodiment, X² is N-oxysuccinimide. This group is suitable forattachment to an amine on a peptide. L may be a straight chain alkylwith 1 to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀)e.g. (CH₂)₄ or (CH₂)₃. L typically has formula -L³-L²-L¹, in which L¹ iscarbonyl, L² is a straight chain alkyl with 1 to 10 carbon atoms (e.g.C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀) e.g. (CH₂)₄ or (CH₂)₅ or L² isabsent, and L³ is —NHC(O)—, carbonyl or —O(CH₃)—.

In one embodiment, L¹ is carbonyl, L² is (CH₂)₅ and L³ is —NHC(O)—. Inanother embodiment, L¹ is carbonyl, L² is (CH₂)₄ and L³ is carbonyl. Inanother embodiment, L¹ is carbonyl, L² is absent and L³ is —O(CH3)-.

In one embodiment, X¹ is

In another embodiment, X¹ is:

In another embodiment, X¹ is

In one embodiment, a compound having the formula X¹-L-X² is

In one embodiment, a compound having the formula X¹-L-X² is

In one embodiment, a compound having the formula X¹-L-X² is:

When the R² group includes a fluorophore, suitable fluorophore groupsmay be prepared according to techniques well known in the art. Forexample as shown in Scheme I, a general protocol is exemplified forpreparing a fluorophore functionalized modifying group.

Target Proteins

A target compound can be one that is a substrate for microbialtransglutaminase, for example proteins that are substrates for microbialtransglutaminase. In one aspect, the target compound contains at leastone Lys residue, and in some embodiments at least two Lys residues. If atarget compound is not a transglutaminase substrate, per se, it ispossible to insert one or more Gln or Lys residues, and in particularLys residues in the protein to make the protein a substrate fortransglutaminase. Alternatively, a peptide sequence containing a lysineresidue (a peptidic tag) may be inserted. In principle, such Gln or Lysresidue may be inserted at any position in the sequence. Typically, theinsertion should be at an accessible portion of the protein or in aflexible loop. It can also be inserted at a position where thephysiological, such as the therapeutic activity of the protein is notaffected to a degree where the protein is not useful anymore, e.g. in atherapeutic intervention. Insertions of amino acid residues in proteinscan be brought about by standard techniques known to persons skilled inthe art, such as post-translational chemical modification ortransgenetic techniques.

Any target compound or protein that is a substrate to transglutaminasecan be modified by the methods disclosed herein, such as e.g. enzymes,protein hormones, growth factors, antibodies and antibody fragments,cytokines, receptors, lymphokines and vaccine antigens. In someembodiments, the polypeptide is an antigenic peptide.

In some embodiments, particularly when R is a polysaccharide, thepolypeptide is a carrier molecule. In general, covalent conjugation ofpolysaccharides to carriers enhances the immunogenicity ofpolysaccharides as it converts them from T-independent antigens toT-dependent antigens, thus allowing priming for immunological memory.Conjugation is particularly useful for pediatric vaccines, (see forexample Ramsay et al. Lancet 357(9251): 195-196 (2001)) and is awell-known technique (see reviews in Lindberg Vaccine 17 Suppl 2:S28-36(1999), Buttery & Moxon, J R Coll Physicians Lond 34, 163-168 (2000),Ahmad & Chapnick, Infect Dis Clin North Am 13:113-33, vii (1999),Goldblatt J. Med. Microbiol. 47, 563-567 (1998), European Patent 477508, U.S. Pat. No. 5,306,492, WO98/42721, Dick et al. Conjugate Vaccines(eds. Cruse et al.) Karger, Basel, 10, 48-114 (1989) and HermansonBioconjugate Techniques, Academic Press, San Diego (1996) ISBN:0123423368.

The carrier protein may be a bacterial toxin toxoid. Useful carrierproteins include bacterial toxins or toxoids, such as diphtheria toxoidor tetanus toxoid, diphtheria and cholera toxins and their subunits suchas fragment C of tetanus toxoid and CRM197 mutant of diphtheria toxin.Other suitable carrier proteins include the N. meningitidis outermembrane protein, synthetic peptides, heat shock proteins, pertussisproteins, cytokines, lymphokines, hormones, growth factors, human serumalbumin (including recombinant), artificial proteins comprising multiplehuman CD4+ T cell epitopes from various pathogen-derived antigens, suchas N19, protein D from H. influenzae, pneumococcal surface protein PspA,pneumolysin, iron-uptake proteins, toxin A or B from C. difficile,recombinant Pseudomonas aeruginosa exoprotein A (rEPA), a GBS protein,etc. In some embodiments, the target protein is a pilus protein such asa GBS protein, for example GBS67 and GBS80.

Further Derivitization

A need for modifying the target proteins of the present invention (i.e.proteins of interest) may arise for any number of reasons, and this isalso reflected in the kinds of compounds that may be selectivelymodified according to the methods of the present invention.

Generally, the methods of the invention comprise a microbialtransglutaminase catalyzed reaction of a protein containing at least twolysines with a glutamine containing peptide of the formula (I)R¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) or (II)R¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z).

In one embodiment, the method consists of the following steps: (a)preparation by peptide synthesis of a compound of the formula (I)R¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z), and purification as known inthe art; (b) mixing excess of this compoundR¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) with a target proteincontaining at least one lysine, and in some embodiments, more than onelysines in an aqueous buffer, optionally containing an organic solvent,detergent or other modifier; (c) addition to this mixture of a catalyticamount of microbial transglutaminase; (d) a mTGase inhibitor canoptionally be added to the mixture; (e) the mixture is subjected to apurification process, typically comprising unit operation such as ultra-or dia-filtration and/or chromatography (ion exchange, size exclusion,hydrophobic interaction, etc.). Selectively modified protein is therebyobtained. The protein is characterized by standard protein analyticalmethods, including chromatography, electrophoresis, peptide mapping andmass spectroscopy.

In one embodiment, the method consists of the following steps: (a)preparation by peptide synthesis of a compound of the formula (II)R¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z), and purification as known inthe art; (b) mixing excess of this compoundR¹-(Leu)_(x)-Gln-(Gly)_(y)-(NH—W—R²)_(z) with a target proteincontaining at least one lysine, and in some embodiments, more than onelysines in an aqueous buffer, optionally containing an organic solvent,detergent or other modifier; (c) addition to this mixture of a catalyticamount of microbial transglutaminase; (d) a mTGase inhibitor canoptionally be added to the mixture; (e) the mixture is subjected to apurification process, typically comprising unit operation such as ultra-or dia-filtration and/or chromatography (ion exchange, size exclusion,hydrophobic interaction, etc.). Selectively modified protein is therebyobtained. The protein is characterized by standard protein analyticalmethods, including chromatography, electrophoresis, peptide mapping andmass spectroscopy.

Optionally, following steps (b) or (c), the modified protein can befurther modified via the functional groups of R¹ or R² or both, ifpresent, for example with a fluorophore label (unless one is alreadypresent). If a label is added, the modified protein may be detectedusing a variety of techniques depending on the nature of the label suchfluorescence or radiolabelling.

In some embodiments, the functional groups of R¹ or R² or both can beradiolabelled. For example, iodine radio labeling can be added to alinker as shown in Scheme II.

As part of the mechanism of the mTGase-mediated transamidation, anintermolecular thioester is formed by reaction between a Cys in theactive site of the MTGase and the Gln substrate. The term“transamidation” is intended to indicate a reaction where nitrogen inthe side chain of glutamine is exchanged with nitrogen from anothercompound, in particular nitrogen from another nitrogen containingnucleophile. This intermediate may be regarded as an activatedGln-residue, the active species being a mTGase-thioester, which reactswith amines, e.g. a protein lysine residue. The selectivity of thereaction is a consequence of 1) the shear steric bulk of themTGase-thioester while interacting with the Lys-bearing proteinsubstrate, and 2) more defined non-covalent interactions between themTGase-thioester and the Lys-bearing protein substrate. An immediateconsequence of this is that proteins carrying activated acyl groups,Acyl-X-protein, where is X is an atom or group that activates the acylgroup towards nucleophilic attack by a protein-lysine amine, is includedin the invention.

Thus, it may be desirable to modify proteins to alter thephysico-chemical properties of the protein, such as e.g. to increase (orto decrease) solubility to modify the bioavailability of therapeuticproteins. In another embodiment, it may be desirable to modify theclearance rate in the body by conjugating compounds to the protein whichbinds to plasma proteins, such as e.g. albumin, or which increase thesize of the protein to prevent or delay discharge through the kidneys.Conjugation may also alter and in particular decrease the susceptibilityof a protein to hydrolysis, such as e.g. in vivo proteolysis.

In another embodiment, it may be desirable to conjugate a label tofacilitate analysis of the protein. Examples of such labels includeradioactive isotopes, fluorescent markers such as the fluorophoresalready described and enzyme substrates.

In still another embodiment, a compound is conjugated to a protein tofacilitate isolation of the protein. For example, a compound with aspecific affinity to a particular column material may be conjugated tothe protein. It may also be desirable to modify the immunogenicity of aprotein, e.g. by conjugating a protein so as to hide, mask or eclipseone or more immunogenic epitopes at the protein. The term “conjugate” asa noun is intended to indicate a modified peptide, i.e. a peptide with amoiety bonded to it to modify the properties of said peptide. As a verb,the term is intended to indicate the process of bonding a moiety to apeptide to modify the properties of said peptide.

In one embodiment, the invention provides a method of improvingpharmacological properties of target proteins. The improvement is withrespect to the corresponding unmodified protein. Examples of suchpharmacological properties include functional in vivo half-life,immunogenicity, renal filtration, protease protection and albuminbinding of any specific protein.

In one aspect, modified proteins of the invention may be furthermodified thru further derivatization of R¹, (A-W—B—R²)_(z), and/orNH—W—R². Specifically, R¹ and/or R² may comprise a chemical groupsuitable for further modification. Examples of such furtherfunctionalization include azide-alkyne Huisgen cycloaddition, morecommonly known as click chemistry, if R¹ or R² includes an azide orcyclooctyne group. If R² includes the tosyl sulfone which eliminates toan α,β-unsaturated ketone, conjugate addition could be applied forfurther modification using a nucleophile such as a thiol.

In some embodiments already described above, W may be selected from:dendrimer, polyalkylene oxide, polyalkylene glycol (PAG), polyethyleneglycol (PEG\ polypropylene glycol (PPG), branched PEGs, polyvinylalcohol (PVA, poly-carboxylate, poly-vinylpyrolidone,polydhykne-co-maleic acid anhydride, polystyrene-c-makic acid anhydride,dextrin, carboxymethyl-dextran; serum protein binding-ligands, such ascompounds which bind to albumin, such as fatty acids, C₅-C₂₄ fatty acid,aliphatic diacid (e.g. C₅-C₂₄), a structure (e.g. sialic acidderivatives or mimetics) which inhibit the glycams from binding toreceptors (e.g. asialoglyco-protein receptor and mannose receptor) asmall organic molecule containing moieties that alters physiologicalconditions, alters charge properties, such as carboxylic acids oramines, or neutral substituents that prevent glycan specific recognitionsuch as smaller alkyl substituents (e.g. C₁-C₅ alkyl), a low molecularorganic charged radical (e.g. C₁-C₂₅), which may contain one or morecarboxylic acids, amines, sulfonic, phosphonic acids, or combinationthereof; a low molecular neutral hydrophilic molecule (e.g. C₁-C₂₅),such as cyclodextrin, or a polyethylene chain which may optionallybranched; polyethyleneglycol with an average molecular weight of 2-40KDa; a well-defined precision polymer such as a dendrimer with an exactmolecular mass ranging from 700 to 20,000 Da, or more preferably between700-10.000 Da; and a substantially non-imunogenic polypeptide such asalbumin or an antibody or part of an antibody optionally containing aFc-domain.

In one embodiment, W is a linear or branched polyethylene glycol havinga molecular weight of between about 40 and about 10,000 amu, alsoreferred to as a “PEG.” The term “PEG” is intended to indicatepolyethylene glycol including analogues thereof, for example where abranching terminal OH-group has been replaced by an alkoxy group, suchas methoxy group, an ethoxy group, or a propoxy group.

Due to the process for producing mPEG these molecules often have adistribution of molecular weights. This distribution is described by thepolydispersity index. The term “polydispersity index” as used hereinmeans the ratio between the weight average molecular weight and thenumber average molecular weight, as known in the art of polymerchemistry (see e.g. “Polymer Synthesis and Characterization”, J. A.Nairn, University of Utah, 2003). The polydispersity index is a numberwhich is greater than or equal to one, and it may be estimated from GelPermeation Chromatographic data. When the polydispersity index is 1, theproduct is monodisperse and is thus made up of compounds with a singlemolecular weight. When the polydispersity index is greater than 1 it isa measure of the polydispersity of that polymer, i.e. the breadth of thedistribution of polymers with different molecular weights.

The use of for example “mPEG2000” in formulas, compound names or inmolecular structures indicates an mPEG residue wherein mPEG ispolydisperse and has a molecular weight of approximately 2,000 Da.

The polydispersity index typically increases with the molecular weightof the PEG or mPEG. When reference is made to 2,000 Da PEG and inparticular 2,000 Da mPEG it is intended to indicate a compound (or infact a mixture of compounds) with a polydisperisty index below 1.06,such as below 1.05, such as below 1.04, such as below 1.03, such asbetween 1.02 and 1.03. When reference is made to 3,000 Da PEG and inparticular 3,000 Da mPEG it is intended to indicate a compound (or infact a mixture of compounds) with a polydispersity index below 1.06,such as below 1.05, such as below 1.04, such as below 1.03, such asbetween 1.02 and 1.03.

In some embodiments, the above methods also include a step ofcontrolling the pH environment of the protein to a pH greater than 7 andcontacting the site selective labeled protein with a peptide having acysteine residue. In some embodiments, the peptide having a cysteineresidue isN⁵—((R)-1-((carboxymethyl)amino)-3-mercapto-1-oxopropan-2-yl)-L-glutamine.In some embodiments, the peptide having a cysteine reside can besubstituted with any thiol containing molecule such as polysaccharideswith thiols, cytotoxics with thiols, thiol-functionalized PEGs, and thelike.

Pharmaceutical Compositions

In another aspect, pharmaceutical compositions comprising a proteinmodified by any of the methods disclosed herein. In one aspect, such apharmaceutical composition comprises a modified protein such as growthhormone (GH), which is present in a concentration from 10-15 mg/ml to200 mg/ml, such as e.g. 10-10 mg/ml to 5 mg/ml and wherein thecomposition has a pH from 2.0 to 10.0. The composition may furthercomprise a buffer system, preservative(s), tonicity agent(s), chelatingagent(s), stabilizers and surfactants. In one embodiment, thepharmaceutical composition is an aqueous composition. Such compositionstypically exist as a solution or a suspension. In a further embodiment,the pharmaceutical composition is an aqueous solution. The term “aqueouscomposition” is defined as a composition comprising at least 50% w/wwater. Likewise, the term “aqueous solution” is defined as a solutioncomprising at least 50% w/w water, and the term “aqueous suspension” isdefined as a suspension comprising at least 50% w/w water.

In another embodiment, the pharmaceutical composition is a freeze-driedcomposition, to which a physician, patient, or pharmacist adds solventsand/or diluents prior to use. In another embodiment the pharmaceuticalcomposition is a dried composition (e.g. freeze-dried or spray-dried)ready for use without any prior dissolution.

In a further aspect, a pharmaceutical composition comprising an aqueoussolution of a modified protein, such as e.g. a Modified GH protein, anda buffer, wherein the modified protein, such as e.g. Modified GH proteinis present in a concentration from 0.1-100 mg/ml or above, and whereinsaid composition has a pH from about 2.0 to about 10.0.

In a another embodiment, the pH of the composition is selected from thelist consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,and 10.0.

In a further embodiment, the buffer is selected from ammoniumbicarbonate, sodium acetate, sodium carbonate, citrate, glycylglycine,histidine, glycine, lysine, arginine, sodium dihydrogen phosphate,disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)aminomethane, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid, TRIS, ormixtures thereof.

In a further embodiment, the composition may also include apharmaceutically acceptable preservative. For example, the preservativemay be phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate,propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal,bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate,chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride,chlorphenesine (3p-chlorphenoxypropane-1,2-diol), or mixtures thereof.The preservative may be present in a concentration from 0.1 mg/ml to 20mg/m or 0.1 mg/ml to 5 mg/ml. In a further embodiment, the preservativeis present in a concentration from 5 mg/ml to 10 mg/ml or from 10 mg/mlto 20 mg/ml.

In a further embodiment, the composition may include an isotonic agent.In a further embodiment, the isotonic agent is selected from a salt(e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such asmono-, di-, or polysaccharides, or water-soluble glucans, including forexample fructose, glucose, mannose, sorbose, xylose, maltose, lactose,sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, solublestarch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.In one embodiment the sugar additive is sucrose. Sugar alcohol isdefined as a C₄-C₈ hydrocarbon having at least one —OH group andincludes, for example, mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, arabitol, and mixtures of the same. In oneembodiment, the sugar alcohol additive is mannitol. In one embodiment,the sugar or sugar alcohol concentration is between about 1 mg/ml andabout 150 mg/ml, or from 1 mg/ml to 50 mg/ml. The isotonic agent ispresent in a concentration from 1 mg/ml to 7 mg/ml or from 8 mg/ml to 24mg/ml, or from 25 mg/ml to 50 mg/ml. The use of an isotonic agent inpharmaceutical compositions is well-known to the skilled person. Forconvenience, reference is made to Remington: The Science and Practice ofPharmacy, 201h edition, 2000.

In the present context, the term “pharmaceutically acceptable salt” isintended to indicate salts which are not harmful to the patient. Suchsalts include pharmaceutically acceptable acid addition salts,pharmaceutically acceptable metal salts, ammonium and alkylated ammoniumsalts. Acid addition salts include salts of inorganic acids as well asorganic acids. Representative examples of suitable inorganic acidsinclude hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric,nitric acids and the like. Representative examples of suitable organicacids include formic, acetic, trichloroacetic, trifluoroacetic,propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic,malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic,methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic,bismethylene salicylic, ethanedisulfonic, gluconic, citraconic,aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic,benzenesulfonic, p-toluenes-ulfonic acids and the like. Further examplesof pharmaceutically acceptable inorganic or organic acid addition saltsinclude the pharmaceutically acceptable salts listed in J. Phann. Sci.1977, 66, 2, which is incorporated herein by reference. Examples ofmetal salts include lithium, sodium, potassium, magnesium salts and thelike. Examples of ammonium and alkylated ammonium salts includeammonium, methylammonium, dimethylammonium, trimethylammonium,ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium,tetramethylammonium salts and the like.

In a further embodiment, the composition includes a chelating agent. Thechelating agent is selected from salts of ethylenediaminetetraaceticacid (EDTA), citric acid, and aspartic acid, and mixtures thereof. Thechelating agent is present in a concentration from 0.1 mg/ml to 5 mg/ml,from 0.1 mg/ml to 2 mg/ml, or from 2 mg/ml to 5 mg/ml. The use of achelating agent in pharmaceutical compositions is well-known to theskilled person. For convenience, reference is made to Remington: TheScience and Practice of Pharmacy, 20^(th) edition, 2000.

In a further embodiment, the composition includes a stabilizer. The useof a stabilizer in pharmaceutical compositions is well-known to theskilled person. For convenience, reference is made to Remington: TheScience and Practice of Pharmacy, 20th edition, 2000. More particularly,compositions of the invention are stabilized liquid pharmaceuticalcompositions whose therapeutically active components include a proteinthat possibly exhibits aggregate formation during storage in liquidpharmaceutical compositions. By “aggregate formation” is intended aphysical interaction between the protein molecules that results information of oligomers, which may remain soluble, or large visibleaggregates that precipitate from the solution. By “during storage” isintended a liquid pharmaceutical composition or composition onceprepared, is not immediately administered to a subject. Rather,following preparation, it is packaged for storage, either in a liquidform, in a frozen state, or in a dried form for later reconstitutioninto a liquid form or other form suitable for administration to asubject. By “dried form” is intended the liquid pharmaceuticalcomposition or composition is dried either by freeze drying (i.e.,lyophilization; see, for example, Williams and Polli (1984) J.Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) inSpray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez,U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Phann.18:1169-1206; and Mumenthaler et al. (1994) Phann. Res. 11:12-20), orair drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser(1991) Biopharm. 4:47-53). Aggregate formation by a protein duringstorage of a liquid pharmaceutical composition can adversely affectbiological activity of that protein, resulting in loss of therapeuticefficacy of the pharmaceutical composition. Furthermore, aggregateformation may cause other problems such as blockage of tubing,membranes, or pumps when the protein-containing pharmaceuticalcomposition is administered using an infusion system.

The pharmaceutical compositions may also include an amount of an aminoacid base sufficient to decrease aggregate formation by the proteinduring storage of the composition. By “amino acid base” is intended anamino acid or a combination of amino acids, where any given amino acidis present either in its free base form or in its salt form. Where acombination of amino acids is used, all of the amino acids may bepresent in their free base forms, all may be present in their saltforms, or some may be present in their free base forms while others arepresent in their salt forms. In one embodiment, amino acids to use inpreparing the compositions of the invention are those carrying a chargedside chain, such as arginine, lysine, aspartic acid, and glutamic acid.Any stereoisomer (i.e., L or D isomer, or mixtures thereof) of aparticular amino acid (methionine, histidine, arginine, lysine,isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof)or combinations of these stereoisomers or glycine or an organic basesuch as but not limited to imidazole, may be present in thepharmaceutical compositions so long as the particular amino acid ororganic base is present either in its free base form or its salt form.In one embodiment the L-stereoisomer of an amino acid is used. In oneembodiment the L-stereoisomer is used. Compositions of the invention mayalso be formulated with analogues of these amino acids. By “amino acidanalogue” is intended a derivative of the naturally occurring amino acidthat brings about the desired effect of decreasing aggregate formationby the protein during storage of the liquid pharmaceutical compositionsof the invention. Suitable arginine analogues include, for example,aminoguanidine, ornithine and N-monoethyl L-arginine, suitablemethionine analogues include ethionine and buthionine and suitablecysteine analogues include S-methyl-L-cysteine. As with the other aminoacids, the amino acid analogues are incorporated into the compositionsin either their free base form or their salt form. In a furtherembodiment, the amino acids or amino acid analogues are used in aconcentration, which is sufficient to prevent or delay aggregation ofthe protein.

In a further embodiment, methionine (or other sulphuric amino acids) oranalogous amino acids, may be added to inhibit oxidation of methionineresidues to methionine sulfoxide when the protein acting as thetherapeutic agent is a protein comprising at least one methionineresidue susceptible to such oxidation. By “inhibit” is intended minimalaccumulation of methionine oxidized species over time. Inhibitingmethionine oxidation results in greater retention of the protein in itsproper molecular form. Any stereoisomer of methionine (L or D isomer) orany combinations thereof can be used. The amount to be added should bean amount sufficient to inhibit oxidation of the methionine residuessuch that the amount of methionine sulfoxide is acceptable to regulatoryagencies. Typically, this means that the composition contains no morethan about 10% to about 30% methionine sulfoxide. Generally, this can beobtained by adding methionine such that the ratio of methionine added tomethionine residues ranges from about 1:1 to about 1000:1, such as 10:1to about 100:1.

In a further embodiment, the composition may include a stabilizerselected from the group of high molecular weight polymers or lowmolecular compounds. The stabilizer may be selected from polyethyleneglycol (e.g. PEG 3350), polyvinyl alcohol (PV A), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride).

The pharmaceutical compositions may also include additional stabilizingagents, which further enhance stability of a therapeutically activeprotein therein. Stabilizing agents include, but are not limited to,methionine and EDTA, which protect the protein against methionineoxidation, and a nonionic surfactant, which protects the protein againstaggregation associated with freeze-thawing or mechanical shearing.

In a further embodiment, the composition also includes a surfactant. Thesurfactant may be selected from a detergent, ethoxylated castor oil,polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fattyacid esters, polyoxypropylenepolyoxyethylene block polymers (e.g.poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100),polyoxyethylene sorbitan fatty acid esters, polyoxyethylene andpolyethylene derivatives such as alkylated and alkoxylated derivatives(tweens, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglyceridesor ethoxylated derivatives thereof, diglycerides or polyoxyethylenederivatives thereof, alcohols, glycerol, lectins and phospholipids (e.g.phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin),derivates of phospholipids (e.g. dipalmitoyl phosphatidic acid) andlysophospholipids (e.g. palmitoyl lysophosphatidyl-L-serine and1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, andglycerophospholipids (e.g. cephalins), glyceroglycolipids (e.g.galactopyransoide), sphingoglycolipids (e.g. ceramides, gangliosides),dodecylphosphocholine, hen egg lysolecithin, fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof₆-C₁₂ (e.g. oleic acid and caprylic acid),acylcarnitines and derivatives, N^(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N^(α)-acylated derivatives of diproteins comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N^(α)-acylated derivative of a triprotein comprising anycombination of a neutral amino acid and two charged amino acids, DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulphate or sodium lauryl sulphate),sodium caprylate, cholic acid or derivatives thereof, bile acids andsalts thereof and glycine or taurine conjugates, ursodeoxycholic acid,sodium cholate, sodium deoxycholate, sodium taurocholate, sodiumglycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,amomc (alkyl-arylsulphonates) monovalent surfactants, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationicsurfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammoniumbromide, cetylpyridinium chloride), nonionic surfactants (e.g. Dodecylβ-D-glucopyranoside), poloxamines (e.g. Tetronic's), which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine, or the surfactantmay be selected from the group of imidazoline derivatives, or mixturesthereof.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience, reference is made to Remington: TheScience and Practice of Pharmacy, 20^(th) edition, 2000.

It is possible that other ingredients may be present in thepharmaceutical composition. Such additional ingredients may includewetting agents, emulsifiers, antioxidants, bulking agents, tonicitymodifiers, chelating agents, metal ions, oleaginous vehicles, proteins(e.g., human serum albumin, gelatin or proteins) and a zwitterion (e.g.,an amino acid such as betaine, taurine, arginine, glycine, lysine andhistidine). Such additional ingredients, of course, should not adverselyaffect the overall stability of the pharmaceutical composition.

Pharmaceutical compositions containing a modified protein, such as e.g.a modified GH protein may be administered to a patient in need of suchtreatment at several sites, for example, at topical sites, for example,skin and mucosal sites, at sites which bypass absorption, for example,administration in an artery, in a vein, in the heart, and at sites whichinvolve absorption, for example, administration in the skin, under theskin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions may be through severalroutes of administration, for example, lingual, sublingual, buccal, inthe mouth, oral, in the stomach and intestine, nasal, pulmonary, forexample, through the bronchioles and alveoli or a combination thereof,epidermal, dermal, transdermal, vaginal, rectal, ocular, for examplesthrough the conjunctiva, uretal, and parenteral to patients in need ofsuch a treatment.

Compositions may be administered in several dosage forms, for example,as solutions, suspensions, emulsions, microemulsions, multiple emulsion,foams, salves, pastes, plasters, ointments, tablets, coated tablets,rinses, capsules, for example, hard gelatin capsules and soft gelatincapsules, suppositories, rectal capsules, drops, gels, sprays, powder,aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses,vaginal pessaries, vaginal rings, vaginal ointments, injection solution,in situ transforming solutions, for example in situ gelling, in situsetting, in situ precipitating, in situ crystallization, infusionsolution, and implants.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the Modified GHprotein, increase bioavailability, increase solubility, decrease adverseeffects, achieve chronotherapy well known to those skilled in the art,and increase patient compliance or any combination thereof. Examples ofcarriers, drug delivery systems and advanced drug delivery systemsinclude, but are not limited to, polymers, for example cellulose andderivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylatepolymers, polylactic and polyglycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, liquid crystals and dispersions thereof,L2 phase and dispersions there of, well known to those skilled in theart of phase behavior in lipid-water systems, polymeric micelles,multiple emulsions, self-emulsifying, self-microemulsifying,cyclodextrins and derivatives thereof, and dendrimers.

Compositions are useful in the composition of solids, semisolids, powderand solutions for pulmonary administration of a modified protein, suchas e.g. a Modified GH protein, using, for example a metered doseinhaler, dry powder inhaler and a nebulizer, all being devices wellknown to those skilled in the art.

Therapeutic Uses of the Modified Proteins

To the extent that the unmodified protein is a therapeutic protein, theinvention also relates to the use of the modified proteins in therapy,and in particular to pharmaceutical compositions comprising the modifiedproteins. Thus, as used herein, the terms “treatment” and “treating”mean the management and care of a patient for the purpose of combating acondition, such as a disease or a disorder. The term is intended toinclude the full spectrum of treatments for a given condition from whichthe patient is suffering, such as administration of the active compoundto alleviate the symptoms or complications, to delay the progression ofthe disease, disorder or condition, to alleviate or relief the symptomsand complications, and/or to cure or eliminate the disease, disorder orcondition as well as to prevent the condition, wherein prevention is tobe understood as the management and care of a patient for the purpose ofcombating the disease, condition, or disorder and includes theadministration of the active compounds to prevent the onset of thesymptoms or complications, The patient to be treated is preferably amammal, in particular a human being, but it may also include animals,such as dogs, cats, cows, sheep and pigs. Nonetheless, it should berecognized that therapeutic regimens and prophylactic (preventative)regimens represents separate aspects for the uses disclosed herein andcontemplated by treating physician or veterinarian.

A “therapeutically effective amount” of a modified protein as usedherein means an amount sufficient to cure, alleviate or partially arrestthe clinical manifestations of a given disease and its complications. Anamount adequate to accomplish this is defined as “therapeuticallyeffective amount”. Effective amounts for each purpose will depend one.g. the severity of the disease or injury as well as the weight, sex,age and general state of the subject. It will be understood thatdetermining an appropriate dosage may be achieved using routineexperimentation, by constructing a matrix of values and testingdifferent points in the matrix, which is all within the ordinary skillsof a trained physician or veterinarian.

The methods and compositions disclosed herein provide modified proteinsfor use in therapy. As such, a typical parenteral dose is in the rangeof 10-9 mg/kg to about 100 mg/kg body weight per administration. Typicaladministration doses are from about 0.0000001 to about 10 mg/kg bodyweight per administration. The exact dose will depend on e.g.indication, medicament, frequency and mode of administration, the sex,age and general condition of the subject to be treated, the nature andthe severity of the disease or condition to be treated, the desiredeffect of the treatment and other factors evident to the person skilledin the art. Typical dosing frequencies are twice daily, once daily,bi-daily, twice weekly, once weekly or with even longer dosingintervals. Due to the prolonged half-lives of the active compoundscompared to the corresponding un-conjugated protein, dosing regimen withlong dosing intervals, such as twice weekly, once weekly or with evenlonger dosing intervals is a particular embodiment. Many diseases aretreated using more than one medicament in the treatment, eitherconcomitantly administered or sequentially administered. It is,therefore, contemplated that the modified proteins in therapeuticmethods for the treatment of one of the diseases can be used incombination with one or more other therapeutically active compoundnormally used in the treatment of a disease. It is also contemplatedthat the use of the modified protein in combination with othertherapeutically active compounds normally used in the treatment of adisease in the manufacture of a medicament for that disease.

EXAMPLES General Preparation Methods for Modifying Compounds

Unless otherwise specified, starting materials were generally availablefrom commercial sources such as Aldrich Chemicals Co. (Milwaukee, Wis.),Lancaster Synthesis, Inc. (Windham, N.H.), Acros Organics (Fairlawn,N.J.). Microbial transglutaminase was provided by Ajinomoto NorthAmerica, Inc. (Itasca, Ill.). CRM₁₉₇ (CAS Number 92092-36-9) isavailable from Aldrich Chemicals Co. (Milwaukee, Wis.). Monomethylauristatin F was purchased from Concortis (San Diego, Calif.).Cyclooctyne reagents were purchased from Synaffix (Nijmegen, TheNetherlands). MenA antigenic polysaccharide was supplied by NovartisNV&D.

Modifying compounds were purified by column chromatography (Interchimpuriflash 430) and analyzed by NMR spectroscopy (400 MHz Bruker), LCMS(Waters Acquity UPLC-UV-CAD-MS), and LCUV (Agilent 1200 series UPLC-UV).Labeled CRM₁₉₇ is characterized by LCMS (UPLC-UV-TOF-MS HRMS WatersAcquity UPLC Qtof). Labeled CRM₁₉₇ is purified by amicon filters (3 kDaor 10 kDa MWCO), and/or SEC (General Electric ÄKTA purifier).

Identification of the protein site of modification (site selectivity)was characterized by protein mapping. The mTGase used in the examples ismicrobial transglutaminase from Anjinomoto North America, Inc. (Itasca,Ill.).

The following acronyms used in the examples below have the correspondingmeanings:

MMAF: monomethylauristatin FmTGase: microbial transglutaminaseMWCO: molecular weight cut off

NHS: N-hydroxysuccinimide

BCN-NHS: (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidylcarbonateHATU: (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate)RT: room temperatureRt: retention time

Z-Q-G-NH-(PEG)₃-N₃

Commercially available ZQG (1 g, 2.96 mmol), Amine-PEG-Azide (0.882 mL,4.45 mmol), DIPEA (1.553 mL, 8.89 mmol), and HATU (1.127 g, 2.96 mmol)were added together in DMF and stirred at RT for 16 hrs. The solutionwas loaded directly onto a 55 g C-18 RP column and purified by columnchromatography 5-80% MeCN/Water. Due to the large amount of MeOHrequired to load sample poor peak shape observed. Reduced volume of thedesired peak and reloaded onto the column. The product was purified asecond time by column chromatography 5-80% MeCN/Water. Yield: 300 mg(19% yield).

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.65-1.79 (m, 1H) 1.82-1.95 (m, 1H)2.07-2.17 (m, 2H) 3.14-3.26 (m, 2H) 3.36-3.44 (m, 4H) 3.46-3.56 (m, 8H)3.57-3.62 (m, 2H) 3.68 (d, J=5.81 Hz, 2H) 3.91-4.04 (m, 1H) 5.03 (d,J=2.27 Hz, 2H) 6.77 (br. s., 1H) 7.23-7.34 (m, 2H) 7.34-7.39 (m, 4H)7.55 (d, J=7.58 Hz, 1H) 7.82 (t, J=5.56 Hz, 1H) 8.16 (t, J=5.68 Hz, 1H);HRMS calculated for (C₂₃H₃₅N₇O₈): 537.2547 observed: (M+1) 538.2623.

ZQ-NH-(PEG)₃N₃

Commercially available NH₂-(PEG)₃-N₃ (0.087 mL, 0.318 mmol), Huenig'sBase (0.069 mL, 0.398 mmol), and commercially available ZQ-NHS (100 mg,0.265 mmol) were combined in DMSO and mixed at RT 16 hrs. The reactionwas loaded directly onto a 35 g C-18 column for purification 10-75%MeCN/H₂O. Yield: 90 mg (70% yield).

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.62-1.75 (m, 1H) 1.77-1.91 (m, 1H) 2.08(dt, J=9.14, 5.88 Hz, 2H) 3.11-3.27 (m, 2H) 3.35-3.43 (m, 4H) 3.48-3.56(m, 8H) 3.57-3.62 (m, 2H) 3.94 (td, J=8.31, 5.38 Hz, 1H) 5.01 (s, 2H)6.73 (br. s., 1H) 7.23 (br. s., 1H) 7.28-7.41 (m, 6H) 7.88 (t, J=5.62Hz, 1H); HRMS calculated for (C₂₁H₃₂N₆O₂): 480.2332 observed: (M+1)481.2440.

Cyclooctyne-cyclopropyl-CH₂—OC(O)NH-Q-G

Commercially available QG (92 mg, 0.316 mmol) and Huenig's Base (64.5μL, 0.369 mmol) were dissolved in 2 mL of 1:1 Water:DMSO and warmed to35° C. Commercially available Click-easy™ BCN N-hydroxysuccinimide esterI (50 mg, 0.246 mmol) was dissolved in 2 mL of DMSO and slowly added tothe reaction. The reaction mixed at 40° C. for 1 hr. The product waspurified by column chromatography (20 g C-18, 0-70 MeCN/Water). Producteluted around 50% MeCN. Yield: 46 mg (49% yield).

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.87 (t, J=9.66 Hz, 2H) 1.27 (quin,J=8.53 Hz, 1H) 1.52 (d, J=10.88 Hz, 2H) 1.62-1.75 (m, 1H) 1.81-1.95 (m,1H) 2.04-2.25 (m, 8H) 3.14 (br. s., 1H) 3.49-3.73 (m, 2H) 3.89-3.99 (m,1H) 4.05 (d, J=7.95 Hz, 2H) 6.72 (br. s., 1H) 7.21-7.31 (m, 2H) 7.88(br. s., 1H); HRMS calculated for (C₁₈H₂₅N₃O₆): 379.1743 observed: (M+1)380.1811.

Cyclooctyne-cyclopropyl-CH₂—OC(O)NH-L-Q-G

Leucine-glutamine-glycine peptide was prepared using a peptidesynthesizer. The synthesizer was programmed for theleucine-glutamine-glycine sequence. The resin was recovered and peptidewas removed with TFA. The solution was slowly added to ether (cold) andto precipitate the product. The solution was centrifuged and ether wasdecanted. The solid was dissolved in water and purified by columnchromatography (35 g RP C-18 column) Product eluted with 100% water in84% yield.

Leucine glutamine glycine peptide (30 mg, 0.07 mmol), Huenig's Base(0.030 mL, 0.174 mmol), and commercially available Click-easy™ BCNN-hydroxysuccinimide ester I (20 mg, 0.070 mmol) were combined in DMFand stirred for five hours at room temp. The reaction was loadeddirectly onto 20 g C-18 column (0-20% MeCN/Water) and purified over 10CV to yield 20 mg (58% yield) of the product. HRMS calculated for(C₂₁H₃₂N₆O₇): 492.2584 observed: (M+1) 493.2682.

Z-Q-NH-(PEG)₂-NHC(O)O—CH₂-cyclopropylcycloctyne

Commercial BocNH-(PEG)3-amine (0.308 mL, 0.994 mmol) in DMSO was addedto commercially available ZQ-NHS (250 mg, 0.663 mmol) and stirred at RTfor 3 hours. The reaction was loaded directly on to a 30 g C-18 columnfor purification (0-50% MeCN/Water). The product eluted at 45% MeCN.Solvent was removed to yield a white residue (200 mg, 59.1% yield). LCMScalculated for (C₂₁H₃₂N₆O₇): 510.27 observed: (M+1) 511.4.

The Boc-protected product (200 mg, 0.392 mmol) was treated with TFA (3mL, 38.9 mmol) and shaken at RT for 10 minutes. The reaction was driedon high vacuum overnight and crude ZQ-NH-(PEG)₂-NH₂ was used directly innext reaction. Huenig's Base (2 mL, 11.45 mmol) was added toZQ-NH-(PEG)₂-NH₂ (150 mg, 0.365 mmol) in 1 mL of DMSO. Commerciallyavailable BCN-NHS (106 mg, 0.365 mmol) was then added and reactionstirred for several hours. Product was purified by column chromatography(35 g C-18 column 15-75% MeCN/Water). Product eluted under DMSO peak aswell as at ˜60% MeCN. Combined fractions and concentrated to 10 mL. Ransecond column at 20-50% MeCN/water. Product eluted at 45% MeCN. Thesolvent was removed under reduced pressure to yield 50 mg (23% yield) ofproduct.

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.78-0.91 (m, 2H) 1.26 (quin, J=8.53 Hz,1H) 1.41-1.59 (m, 2H) 1.62-1.75 (m, 1H) 1.77-1.90 (m, 1H) 1.95-2.30 (m,8H) 3.11 (q, J=5.95 Hz, 2H) 3.20 (td, J=12.87, 6.66 Hz, 2H) 3.36-3.42(m, 4H) 3.49 (s, 4H) 3.88-3.98 (m, 1H) 4.02 (d, J=8.07 Hz, 2H) 5.01 (s,2H) 6.73 (br. s., 1H) 7.07 (t, J=5.44 Hz, 1H) 7.23 (br. s., 1H)7.29-7.37 (m, 6H) 7.88 (t, J=5.62 Hz, 1H). HRMS calculated for(C₃₀H₄₂N₄O₈): 586.3003 observed: (M+1) 587.3092.

Z-Q-NH—(CH₂)₃-dimethylacetal

ZQNHS (100 mg, 0.265 mmol), 4,4-dimethoxybutan-1-amine (0.049 mL, 0.292mmol), and Huenig's Base (0.046 mL, 0.265 mmol) were dissolved in DMFand mixed at RT for 1 hr. The reaction was loaded directly onto a 35 gC-18 column for purification (0-40% MeCN/H₂O). The solvent was removedunder reduced pressure to yield 50 mg (48% yield) of the product.

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.30-1.43 (m, 2H) 1.43-1.54 (m, 2H)1.58-1.75 (m, 1H) 1.76-1.89 (m, 1H) 2.01-2.16 (m, 2H) 3.04 (dt, J=12.57,6.47 Hz, 2H) 3.19 (s, 6H) 3.89 (td, J=8.46, 5.31 Hz, 1H) 4.32 (t, J=5.56Hz, 1H) 5.00 (s, 2H) 6.76 (br. s., 1H) 7.26 (br. s., 1H) 7.28-7.42 (m,6H) 7.86 (t, J=5.68 Hz, 1H) LCMS calculated for (C₁₉H₂₉N₃O₆): 395.21observed: (M+1) 396.5.

Z-Q-NH—(CH₂)₂—NH—C(O)—CH₂-Alexafluor647

Commercially available ZQ-NHS (286 mg, 0.758 mmol), Huenig's Base (0.5mL, 2.86 mmol) and tert-butyl(2-aminoethyl)carbamate (0.3 mL, 1.498mmol) were combined in DCM and sonicated. The solution stirred 16 hours.The reaction was filtered and washed with DCMm followed by evaporation.The residue was dissolved in MeOH/DCM 1:10, and passed through a HCO₃catch and release column to remove the acid side product. The resultingproduct was treated with TFA (1 mL, 12.98 mmol) in 15 mL of DCM wereadded. Reaction mixed for 30 minutes and then evaporated on rotovap. Theresidue was dissolved in MeOH/DCM 1:10 and passed through a HCO₃ catchand release column to remove acid side product. The solvent was removedto yield 300 mg (91% yield) of the product.

The resulting amine linker (5.1 mg, 0.016 mmol) was dissolved in DMSO(0.5 mL) and Huenig's Base (3.66 μl, 0.021 mmol) was added followed byAlexflour647 (5 mg, 5.24 μmol). The reaction stirred at room temp for X.The reaction was loaded directly onto a 35 g C-18 column for columnchromatography purification (5-35% MeCN/Water). Product eluted ˜10%MeCN. The product was lyophilized to yield a dark purple powder. Yield:3.5 mg (57% yield). HRMS calculated for (C₅₁H₆₇N₆O₁₇S₄ ⁺): massexpected: 1163.34 mass observed: M+1, 1164.

Z-Q-NH—(CH₂)₂—C(O)—(CH₂)₂—SO₂-Tol

3-((tert-butoxycarbonyl)amino)propanoic acid (2 g), HATU (4.42 g),Huenig's Base (4.62 mL) and N,O-dimethylhydroxylamine hydrochloride(1.134 g) were combined in DCM and stirred at RT for two hours. Thereaction was poured into water and the organics were extracted. Theorganic layer was evaporated to yield crude yellow oil. The product waspurified by column chromatography (50 g C-18, 10-70% MeCN/Water).Fractions collect and concentrated to yield a yellow oil Yield: 1.3 g,53% yield. Expected Mass: 232, Observed M+1: 233.

tert-Butyl (3-(methoxy(methyl)amino)-3-oxopropyl)carbamate (1.3 g, 5.60mmol) was dissolved in dry THF and the flask was purged with nitrogen.The solution was cooled to 0° C. then vinylmagnesium bromide (20 ml,20.00 mmol) in THF was slowly added. The reaction warmed to RT ON. Thereaction was poured into cold sat. NH₄Cl and extracted with ethylacetate. The organics were dried and the solvent was removed on rotovap.The crude material was carried on.

tert-Butyl (3-oxopent-4-en-1-yl)carbamate and 4-methylbenzenethiol werecombined in MeOH and stirred at RT 3 days. Methanol was removed byrotovap to yield a thick oily yellow residue. The residue was dissolvedin DCM and washed with water 2×. The organic layer was dried and thesolvent removed on rotovap to yield a yellow oil. This product (1 g,3.09 mmol) was dissolved in DCM and cooled to 0° C. TFA (3 mL, 38.9mmol) was slowly added then warmed to RT and stirred for 1 hour. Solventwas removed and residue was dissolved in DCM and passed through acarbonate catch and release column followed by a carboxylic acid catchand release. Product went through both. Columns were washed, the solventwas combined and then solvent by rotovap. Product was purified by columnchromatography (50 g C-18 10-70% MeCN/Water). The product was collectedsolvent removed by evaporation to yield a yellow oil. The product wascollected and the solvent was removed by evaporation to yield a yellowoil. The product was carried on without any further purification. 850mg, quantitative yield.

1-Amino-5-(p-tolylthio)pentan-3-one (100 mg, 0.448 mmol) and Huenig'sBase (0.130 mL, 0.746 mmol) were dissolved in DMF then commerciallyavailable ZQ-NHS (141 mg, 0.373 mmol) was added and mixed at RT. Theproduct was purified by column chromatography (25 g C-18 column 10-75%MeCN/Water). The product was collected and organics evaporated byrotovap. MeOH was added to the sulfide linker in water. Oxone (229 mg,0.373 mmol) was added and the reaction stirred at RT ON. The reactionwas filtered and purified by column chromatography (25 g C-18 column10-75% MeCN/Water) and product was lyophilized to 75 mg (39% yield).

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.59-1.72 (m, 1H) 1.75-1.89 (m, 1H) 2.06(dt, J=8.99, 5.90 Hz, 1H) 2.42 (s, 3H) 2.60 (t, J=6.60 Hz, 2H) 2.77 (t,J=7.40 Hz, 2H) 3.11-3.26 (m, 2H) 3.39-3.48 (m, 2H) 3.86 (td, J=8.28,5.32 Hz, 1H) 5.01 (d, J=1.83 Hz, 3H) 6.74 (br. s., 1H) 7.23 (br. s., 1H)7.28-7.42 (m, 6H) 7.47 (d, J=8.19 Hz, 2H) 7.78 (d, J=8.19 Hz, 2H) 7.83(t, J=5.50 Hz, 1H). HRMS calculated for (C₂₅H₃₁N₃O₇S): mass expected:517.1883 mass observed: M+1, 518.1974.

Ac-L-Q-G-NH—(CH₂)₂—C(O)—(CH₂)₂—SO₂-Tol

1-Amino-5-(p-tolylthio)pentan-3-one was prepared as described above inthe synthesis of Z-Q-NH—(CH₂)₂—C(O)—(CH₂)₂—SO₂-Tol. LQG was prepared asdescribed above in the synthesis ofCyclooctyne-cyclopropyl-CH₂—OC(O)NH-L-Q-G. LQG was acelated at theN-terminus by combining LQG (27 mg, 0.085 mmol), Ac2O (9.66 μl, 0.102mmol) and Huenig's Base (0.045 mL, 0.256 mmol) in DCM and stirring at RTfor several hours. The solvent was evaporated and water was added. Theproduct was lyophilized and used directly in the next reaction. LCMSObserved Mass: (M+1) 359.2; Desired Mass: 358.4

Ac-LQG (38 mg, 0.106 mmol), 1-Amino-5-(p-tolylthio)pentan-3-one (47.4mg, 0.212 mmol), and Huenig's Base (0.074 mL, 0.424 mmol) were combinedin DMF. HATU (40.3 mg, 0.106 mmol) was added and the reaction stirred atRT ON. Product was purified by column chromatography (35 g C-18 column10-50% MeCN/Water) to yield.

The sulfide (13.5 mg, 0.024 mmol) was dissolved in 1:1 Water:MeOH. Oxone(44.2 mg, 0.072 mmol) was added and the reaction stirred at RT ON. Theproduct was purified by column chromatography (25 g C-18 column 10-60%MeCN/Water).

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.85 (dd, J=16.08, 6.54 Hz, 6H)1.36-1.45 (m, 2H) 1.59 (dt, J=13.27, 6.57 Hz, 1H) 1.69-1.79 (m, 1H) 1.83(s, 3H) 2.00-2.14 (m, 2H) 2.41 (s, 3H) 2.57-2.64 (m, 2H) 2.75 (t, J=7.40Hz, 2H) 3.18 (q, J=6.52 Hz, 1H) 3.27 (s, 2H) 3.41 (t, J=7.40 Hz, 2H)3.59 (dd, J=5.75, 3.18 Hz, 2H) 4.07-4.17 (m, 1H) 4.21-4.30 (m, 1H) 6.74(br. s., 1H) 7.24 (br. s., 1H) 7.46 (d, J=7.95 Hz, 2H) 7.68 (t, J=5.50Hz, 1H) 7.76 (d, J=8.31 Hz, 2H) 7.93-8.05 (m, 2H) 8.11 (d, J=7.09 Hz,1H) HRMS calculated for (C₂₇H₄₁N₅O₈S) Desired Mass: 595.2676 ObservedMass: (M+1) 596.2755.

Z-Q-NH—(CH₂)₄—C(O)—(CH₂)₂—SO₂-Tol

6-((tert-Butoxycarbonyl)amino)hexanoic acid (1 g, 4.32 mmol) wasdissolved in DCM then N,O-dimethylhydroxylamine hydrochloride (0.464 g,4.76 mmol), HATU (1.808 g, 4.76 mmol), and TEA (0.723 mL, 5.19 mmol)were added. The reaction stirred at RT for several hours, reaction notcomplete, so additional amine, HATU, and TEA added. The reaction stirred16 hrs. The reaction was filtered and solvent was evaporated. Ether wasadded and the reaction was filtered again. The product was purified bycolumn chromatography (25 g column 0-45% EtOAc/Hep) to 385 mg (32.5%yield).

Tert-Butyl (6-(methoxy(methyl)amino)-6-oxohexyl)carbamate (385 mg, 1.403mmol) was dissolved in dry THF and cooled to 0° C. Vinylmagnesiumbromide (4.210 mL, 4.21 mmol) was slowly added. The reaction was allowedto warm to RT overnight. The reaction was poured into sat. NH₄Cl andorganics were extracted with EtOAc. The organic layer was washed withwater and brine, dried and evaporated. The product was purified bycolumn chromatography (Sunfire RP HPLC 10-40% MeCN/Water 0.1% TFA over10 minutes 226 nm UV) to give 280 mg (83% yield).

Tert-Butyl (6-oxooct-7-en-1-yl)carbamate (280 mg, 1.160 mmol) and4-methylbenzenethiol (173 mg, 1.392 mmol) were dissolved in MeOH andstirred at RT for 16 hrs. Methanol was removed and the product waspurified by chromatography (25 g column 0-30% EtOAc/Hep) to yield 160 mg(38% yield).

Tert-Butyl (6-oxo-8-(p-tolylthio)octyl)carbamate (160 mg, 0.438 mmol)and ozone (807 mg, 1.313 mmol) were combined together in MeOH/Water50/50 and stirred at RT for 16 hours. The reaction was poured into waterand extracted with DCM. The organic layer was dried by evaporation and 4Molar HCl in dioxane was added and stirred at RT for several hours. Thesolvent was removed by evaporation to yield an off-white residue. Theproduct was purified by a Sunfire RP HPLC (10-40% MeCN/Water 0.1% TFA)to yield 33 mg (25% yield).

8-amino-1-tosyloctan-3-one (20 mg, 0.067 mmol) and commerciallyavailable ZQNHS (23.07 mg, 0.061 mmol) were combined in DMSO and mixedat 37° C. for an hour. Reaction not complete and no progress wasobserved after several hours. Huenig's Base (5.34 μl, 0.031 mmol) wasadded and the reaction advanced. The reaction was loaded directly ontocolumn (6 g C-18 0-75% MeCN/Water) for purification. Column ran twice to2.4 mg (7% yield).

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.17 (q, J=7.58 Hz, 2H) 1.28-1.44 (m,4H) 1.61-1.75 (m, 1H) 1.77-1.90 (m, 1H) 2.00-2.16 (m, 2H) 2.37-2.43 (m,5H) 2.74 (t, J=7.34 Hz, 2H) 2.92-3.08 (m, 2H) 3.43 (t, J=7.34 Hz, 2H)3.90 (td, J=8.31, 5.50 Hz, 1H) 5.01 (s, 2H) 6.74 (br. s., 1H) 7.25 (br.s., 1H) 7.28-7.40 (m, 6H) 7.46 (d, J=7.82 Hz, 2H) 7.77 (d, J=8.31 Hz,2H) 7.78-7.84 (m, 1H). HRMS calculated for (C₂₈H₃₇N₃O₇S): 559.2352observed: (M+1) 560.2426.

Z-Q-NH-MenA Polysaccharide

Amine functionalized MenA antigenic polysaccharide (10 mg) was added tocommercially available ZQNHS (10 mg 26.4 umol) in DMSO with base andstirred for several hours. The reaction was lyophilized, dissolved inwater, and purified by a 10 KD Amicon filter 4×. The flow through wasthen passed through a 3 KD Amicon to recover additional product. Thiswas carried on crude with an estimated yield of ˜50%.

Z-Q-NH—(CH₂)₅—C(O)-monomethylauristatin F

6-((tert-butoxycarbonyl)amino)hexanoic acid (46.5 mg, 0.201 mmol) wasdissolved in DMSO (1 mL) and Huenig's Base (0.08 mL, 0.458 mmol) andHATU (76 mg, 0.201 mmol) were added. The reaction stirred at RT for 30minutes before adding the reaction mixture to MMAF (50 mg, 0.067 mmol).The reaction then stirred at RT for 4 hours. The reaction mixture wasloaded directly onto 35 g C-18 column for purification by columnchromatography (5-75% MeCN/Water 0.1% formic acid). The solvent wasremoved on rotovap and placed on high vac overnight.

The resulting product was treated with TFA (2 mL) at 0° C. and broughtto room temp to stir for 5 min. The reaction was loaded directly onto a35 g C-18 column for column purification. The solvent was removed underreduced pressure to yield 30 mg (52% yield).

The deprotected amine (20 mg, 0.023 mmol) was dissolved in THF then LiOH(1 mL, 4.00 mmol) was added. The reaction stirred at RT for 30 minuteswas then loaded directly onto column (35 g C-18 column) for purification(0% MeCN followed by 20-40% MeCN/Water). Product eluted with THF andcolumn rerun under above conditions to yield 10 mgs in 51% yield.

The resulting product (5 mg, 5.92 μmol) was dissolved in DMSO, added tocommercially available ZQNHS (4.4 mg, 0.012 mmol), and stirred at 37° C.for 20 minutes. The reaction mixture was loaded directly onto a 25 gC-18 column for column chromatography purification (20-75% MeCN/Water).The solvent was removed under reduced pressure to yield 3 mg in 46%yield. LCMS calculated for (C₂₁H₃₂N₆O₂): 1106.6627 observed: (M+1)1107.9.

Phenol-(CH₂)₂—C(O)-L-Q-G

LQG was prepared as described in “Synthesis ofCyclooctyne-cyclopropyl-CH₂—OC(O)NH-L-Q-G” above. Commercially available2,5-dioxopyrrolidin-1-yl 3-(4-hydroxyphenyl)propanoate (14.65 mg, 55.6μmol) was added to LQG (16 mg, 50.6 μmol) in DMSO with Huenig's Base (18uL, 101 μmol) and reaction stirred at room temp overnight. Reactionloaded directly on 20 g column for purification (10-50% MeCN/Water) toyield 13 mg of product for a 55% yield. HRMS calculated for(C₂₁H₃₂N₆O₂): 464.2271 observed: (M+1) 465.2356.

ZQ(PEG)₂azidobenzylamide

4-Azidophenylacetic acid N-succinimido ester (ChemPacific, 26.7 mg.0.073 mmol) was dissolved in DMF (1 mL) and combined with a solution ofbenzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate(20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121 mL, 0.585 mmol) wasadded and the reaction was mixed at r.t. for 4 hours at which pointDIPEA (61 μL, 6 eq.) and 4-azidophenylacetic acid N-succinimido ester (9mg, 0.024 mmol) were added. The reaction was mixed at r.t. for 2 days.4-azidophenylacetic acid N-succinimido ester (89 mg, 0.24 mmol) wasadded and the reaction was stirred at r.t. for 2 hours. The solution waspurified via MS-triggered HPLC (100-Prep3; Acid Method 3; Sunfire 30×50mm 5 μm column ACN/H₂O w/0.1% TFA 75 ml/min, A: Water (0.1% formicacid); B: ACN gradient 0 min 5% B; 5% to 95% B in 1.70 min; 2.0 min 95%B; 2.1 min 5% B flow rate 2 ml/min

1.5 ml injection; Tube Trigger M=570). Fractions with desired productwere pooled and lyophilized to give 3.6 mg of light yellow powder (13%)of ZQ(PEG2)azido-benzylamide (benzyl(17-amino-1-(4-azidophenyl)-2,13,17-trioxo-6,9-dioxa-3,12-diazaheptadecan-14-yl)carbamates).LCMS SQ2; Product Analysis-Acidic; R_(t)=1.79: MS [M+H] observed: 570.3,calculated: 569.6.

ZQ(PEG)₂amidoethylmethyldiazirin

Sulfo-NHS-diazirine (Thermo Scientific, 23.92 mg, 0.073 mmol) wasdissolved in DMF (1 mL) and combined with a solution of benzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate(20 mg, 0.049 mmol) in DMF (2.3 mL). The reactants were stirred tocombine and DIPEA (0.121 mL, 0.585 mmol) was added. The reaction wasstirred at r.t. for 2 hours at which point the reaction became cloudyand 0.5 mL DMF was added. The reaction was stirred for an additional 2hours and DIPEA (61 μL) and sulfo-NHS-diazirine (8 mg) were added. Thereaction was mixed at r.t. for 16 hours at which point the consumptionof starting material was observed by LCMS analysis. The solution waspurified via MH-triggered HPLC (100-Prep3; Acid Method 3; Sunfire 30×50mm Sum column ACN/H₂O w/0.1% TFA 75 ml/min, 1.5 ml injection; TubeTrigger M=521). Fractions with the desired product,ZQ(PEG)₂amidoethylmethyldiazirin (benzyl(18-amino-1-(3-methyl-3H-diazirin-3-yl)-3,14,18-trioxo-7,10-dioxa-4,13-diazaoctadecan-15-yl)carbamate)were pooled and lyophilized to give 2 mg of the desired compound as awhite powder (8%). LCMS SQ2; Product Analysis-Acidic; R_(t)=1.49: MS[M+H] observed: 521.4, calculated: 520.6.

2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-undecyltridecanedioic acid

A solution of DCC (126 mg, 0.610 mmol) in DCM (1.57 mL) was added to asolution of intermediate 4 and N-hydroxysuccinimide in DCM (5 mL) andTHF (5 mL) under N₂. After 3.5 hours, the solvent was evaporated and theresidue purified by supercritical fluid chromatography (SFC; Princeton2-ethyl-pyridine, 20×150 mm, 20-30% MeOH/CO₂), yielding the titlecompound as a colorless oil (138 mg, 0.256 mmol, 50%): LCMS method BRt=1.21 min, M+H 540.5; ¹H NMR (600 MHz, ACETONITRILE-d3) δ ppm 0.91 (t,J=7.20 Hz, 3H) 1.22-1.42 (m, 34H) 1.57 (quin, J=7.34 Hz, 2H) 1.93-1.96(m, 2H) 2.28 (t, J=7.47 Hz, 2H) 2.79 (br. d, J=6.30 Hz, 4H).

2-((2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-5-azatetracontan-40-yl)carbamoyl)-2-undecyltridecanedioicacid

t-Boc-N-amido-dPEG®₁₁-amine (100 mg, 0.155 mmol, Quanta Biodesign) and2-((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-undecyltridecanedioic acid(80 mg, 0.148 mmol) were dissolved in THF (3 mL) and stirred at roomtemperature under nitrogen nitrogen. After 30 minutes, DIPEA (0.05 mL,0.286 mmol) was added and the reaction mixture stirred at roomtemperature overnight. Complete conversion was observed by LCMS (AcidicEluent A: Water+0.05% Trifluoroacetic Acid, Eluent B: ACN, columnSunfire C18 3.5 μm 3.0×30 mm −40° C., 5-95% gradient 2 minutes,retention time 1.92 min). The reaction mixture was concentrated underreduced pressure, then dissolved in about 1.5 mL of acetonitrile.Purified on a MS-triggered HPLC (Sunfire 30×50 mm Sum column ACN/H2Ow/0.1% TFA 75 ml/min 1.5 ml injection, 65-95% ACN 3.5 min gradient,retention time 3.23 minutes) and the fractions pooled and lyophilized togive 85 mg clean product in 54% yield. Clear oil. LCMS: SQ4,RXNMON-Acidic-NonPolar Rt=1.18 mM, M+H 1070.1; ¹H NMR (400 MHz,ACETONITRILE-d₃) δ ppm 0.82-1.03 (m, 1H) 1.11-1.37 (m, 10H) 1.37-1.51(m, 2H) 1.51-1.64 (m, 1H) 1.69-1.82 (m, 1H) 1.90-2.04 (m, 66H) 2.05-2.21(m, 8H) 2.21-2.42 (m, 1H) 3.17-3.28 (m, 1H) 3.40-3.68 (m, 13H).

2-((35-Amino-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)carbamoyl)-2-undecyltridecanedioicacid

2-((2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-5-azatetracontan-40-yl)carbamoyl)-2-undecyltridecanedioicacid (5 mg, 4.68 μmol) was dissolved in DCM (Volume: 2 mL), thentrifluoroacetic acid (25 μl, 0.324 mmol) was added. The reaction mixturewas stirred at room temperature under nitrogen atmosphere for about 2hours. Complete conversion was observed by LCMS (Acidic Eluent A:Water+0.05% Trifluoroacetic Acid, Eluent B: ACN, column Sunfire C18 3.5μm 3.0×30 mm −40° C., 5-95% gradient 2 minutes, retention time 1.45 min)The reaction mixture was concentrated under reduced pressure, thenrinsed with DCM and concentrated again 3 times. Dissolved in a mixtureof acetonitrile and DMSO. Purified on a MS-triggered HPLC (Sunfire 30×50mm Sum column ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection, 45-70% ACN3.5 min gradient, retention time 2.50 minutes) and the fractions pooledand lyophilized to give 2.5 mg clean product in 55% yield. Clear oil.

LCMS ZQ1 RXNMON_Acidic Rt=1.45 min, M+H 969.9; ¹H NMR (400 MHz,ACETONITRILE-d₃) δ ppm 0.62-0.91 (m, 2H) 0.91-1.10 (m, 3H) 1.10-1.31 (m,18H) 1.46 (quin, J=7.21 Hz, 2H) 1.59-1.89 (m, 35H) 1.94-2.09 (m, 1H)2.16 (t, J=7.40 Hz, 2H) 2.97-3.11 (m, 1H) 3.24-3.37 (m, 1H) 3.37-3.61(m, 28H) 3.61-3.89 (m, 2H) 7.85 (br. s., 1H).

2-(((S)-5-(3-Amino-3-oxopropyl)-3,6-dioxo-1-phenyl-2,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-4,7-diazadotetracontan-42-yl)carbamoyl)-2-undecyltridecanedioicacid (ZQ-FA)

A solution of 2-((2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-5-azatetracontan-40-yl)carbamoyl)-2-undecyltridecanedioicacid (20 mg, 0.018 mmol) in THF (Volume: 2 mL) was added to Z-L-Gln-Osu(Santa Cruz Biotechnology, CAS 34078-85-8, 11 mg, 0.029 mmol), thenDIPEA (75 μl, 0.429 mmol) was added. Stirred at room temperature under anitrogen atmosphere over weekend. Complete conversion was observed byLCMS (Acidic Eluent A: Water+0.05% Trifluoroacetic Acid, Eluent B: ACN,column Sunfire C18 3.5 μm 3.0×30 mm −40° C., 5-95% gradient 2 minutes,retention time 1.77 min) The reaction mixture was concentrated underreduced pressure and then dissolved in acetonitrile. Purified on aMS-triggered HPLC (Sunfire 30×50 mm Sum column ACN/H2O w/0.1% TFA 75ml/min 1.5 ml injection, 55-80% ACN 3.5 min gradient, retention time2.70 minutes) and the fractions pooled and lyophilized to give 10.5 mgclean product ZQ-FA in 46% yield as a clear colorless oil.

LCMS SQ4 RXNMON_Acidic Rt=1.60 min, M+H 1232.4; ¹H NMR (400 MHz,ACETONITRILE-d₃) δ ppm 0.67-0.93 (m, 2H) 0.93-1.10 (m, 2H) 1.10-1.32 (m,15H) 1.45 (quin, J=7.24 Hz, 1H) 1.59-1.69 (m, 1H) 1.75-1.93 (m, 30H)1.94-2.21 (m, 20H) 3.23 (quin, J=5.26 Hz, 1H) 3.28-3.51 (m, 23H) 3.95(td, J=7.73, 5.44 Hz, 1H) 4.92-5.22 (m, 1H) 5.78 (br. s., 1H) 6.13-6.42(m, 1H) 6.88 (br. s., 1H) 7.20-7.36 (m, 2H) 7.42 (t, J=5.07 Hz, 1H).

Azido-nitrophenyl-glutamine-glycine

QG (30 mg, 0.148 mmol) was dissolved in DMF (Volume: 1 mL, Ratio: 1.000)and sodium1-((4-azido-2-nitrobenzoyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (60.1mg, 0.148 mmol) was added in H₂O (Volume: 1.000 mL, Ratio: 1.000)followed by addition of DIPEA (0.177 mmol). The reaction stirred for 16hours at which time the product was purified by HPLC (Sunfire 30×50 mm 5um column ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection, rt=1.53) togive the desired product in 62% yield. LCMS SQ4 RXNMON_Acidic Rt=0.68min, M+H 394.3; ¹H NMR (400 MHz, methanol-d₄) 6 ppm 1H NMR (METHANOL-d4,400 MHz): 8.12-8.26 (m, 1H), 7.21-7.40 (m, 2H), 4.60 (dd, J=8.3, 5.7 Hz,1H), 3.83-4.15 (m, 2H), 2.36-2.54 (m, 2H), 2.21 (d, J=7.5 Hz, 1H), 2.07(d, J=6.6 Hz, 1H).

Diazirine-QG

QG (30 mg, 0.148 mmol) was dissolved in DMF (Volume: 1 mL, Ratio: 1.000)and sodium1-((3-(3-methyl-3H-diazirin-3-yl)propanoyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate(50 mg, 0.153 mmol) in H₂O (Volume: 1.000 mL, Ratio: 1.000) was addedfollowed by DIPEA (0.031 mL, 0.177 mmol). Reaction stirred 16 hours atwhich time product was directly purified by HPLC (Sunfire 30×50 mm Sumcolumn 15-20% gradient ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection,rt=2.46) to give the desired product in 52% yield. LCMS SQ4RXNMON_Acidic Rt=0.61 min, M+H 314.2; ¹H NMR (400 MHz, methanol-d₄) δppm 1H NMR (METHANOL-d4, 400 MHz): 4.39 (dd, J=8.3, 5.7 Hz, 1H),3.76-4.04 (m, 2H), 2.34 (m, 2H), 2.11-2.19 (m, 2H), 2.07-2.11 (m, 1H),1.89-2.00 (m, 1H), 1.62-1.74 ppm (m, 2H), 1.01 (s, 3H).

ZQ(PEG)₃ Biotin

ZQ NHS (45.1 mg, 0.119 mmol), biotin amine (Pierce cat #21347, 50 mg,0.119 mmol), and DIPEA (23 uL, 0.131 mmol) were combined in DMF (2 mL)and stirred at room temp for 2 hours at which time LCMS showspredominantly product. Reaction loaded on HPLC (Sunfire 30×50 mm Sumcolumn 15-20% gradient ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection,rt=2.46) to give the desired product in 43% yield. LCMS SQ4RXNMON_Acidic Rt=0.79 min, M+H 681.4; 1H NMR (METHANOL-d4, 400 MHz):d=7.33-7.41 (m, 4H), 7.27-7.33 (m, 1H), 5.08 (s, 2H), 4.48 (dd, J=7.8,4.4 Hz, 1H), 4.29 (dd, J=7.9, 4.5 Hz, 1H), 4.12 (m, 1H), 3.57-3.67 (m,8H), 3.49-3.57 (m, 4H), 3.34-3.43 (m, 4H), 3.15-3.23 (m, 1H), 2.92 (dd,J=12.8, 5.1 Hz, 1H), 2.70 (d, J=12.8 Hz, 1H), 2.26-2.36 (m, 2H), 2.21(t, J=7.4 Hz, 2H), 2.04 (m, 1H), 1.92 (m, 1H), 1.53-1.77 (m, 4H), 1.44ppm (m, 2H).

Conjugation of Modifying Compounds to CRM₁₉₇ Z-Q-G-NH-(PEG)₃-N₃

32 μL of CRM197 (32 mg/mL) is added to 1000 μL of Z-Q-G-NH-(PEG)₃-N₃ (2mg/mL) in 100 mM pH 8 Tris buffer and 100 μL of microbialtransglutaminase (stock of 50 mg/mL in PBS 1× prepared from commercial1% mTGase in maltocyclodextrin) are added. Reaction incubated at 25° C.for 30 minutes. Reaction purified via SEC with a running buffer of PBS1× over 1.5 CV. One addition of the linker is observed by Mass Spectrum.LCMS calculated: 58929; observed: (M+1) 58930. Yield: 700 ug, 68% yield.

32 μL of CRM197 (32 mg/mL) is added to 1000 μL of Z-Q-G-NH-(PEG)₃-N₃ (2mg/mL) in 100 mM pH 8 Tris buffer and 100 μL of microbialtransglutaminase (stock of 50 mg/mL in PBS 1× prepared from commercial1% mTGase in maltocyclodextrin) are added. Reaction incubated at 25° C.for 18 hours. Reaction purified via SEC with a running buffer of PBS 1×over 1.5 CV. Two additions of the linker are observed by Mass Spectrum.LCMS calculated: 59450; observed: (M+1) 59451. Yield: 700 ug, 68% yield.

32 uL of CRM197 (32 mg/mL) is added to 1000 μL, of Z-Q-G-NH-(PEG)₃-N₃ (2mg/mL) in 100 mM pH 6 Sodium acetate buffer and 100 uL of microbialtransglutaminase (stock of 50 mg/mL in PBS 1× prepared from commercial1% mTGase in maltodextrin) is added. Reaction incubated at 25° C. for 3days. Reaction purified via SEC with a running buffer of PBS 1× over 1.5CV. Three and four additions are observed by Mass Spectrum. LCMScalculated: 59971, 60492; observed: (M+1) 59972, 60493. Yield: 700 ug,68% yield.

ZQ-NH-(PEG)₃N₃

63 μL of CRM197 (32 mg/mL) is added to 1800 μL of ZQ-NH-(PEG)₃N₃ (2mg/mL) in 100 mM pH 8 Tris buffer and 150 uL of microbialtransglutaminase (stock of 50 mg/mL in PBS 1× prepared from commercial1% mTGase in maltocyclodextrin) are added. Reaction incubated at 25° C.for 1 hour. Reaction purified via SEC with a running buffer of PBS 1×over 1.5 CV. One addition of the linker is observed by Mass Spectrum.LCMS calculated: 58872; observed: (M+1) 58875. Yield: 1.3 mg (67%).

Cyclooctyne-cyclopropyl-CH₂—OC(O)NH-Q-G

32 μL of CRM197 (32 mg/mL) is added to 1000 μL, ofCyclooctyne-cyclopropyl-CH₂—OC(O)NH-Q-G (2 mg/mL) in 100 mM pH 8 Trisbuffer and 100 μL of microbial transglutaminase (stock of 50 mg/mL inPBS 1× prepared from commercial 1% mTGase in maltocyclodextrin) areadded. Reaction incubated at 25° C. for 3 hours. Reaction purified viaSEC with a running buffer of PBS 1× over 1.5 CV

One addition of the linker is observed by Mass Spectrum. LCMScalculated: 58771 observed: (M+1) 58771. Yield: 0.475 mg, 50% yield.

Cyclooctyne-cyclopropyl-CH₂—OC(O)NH-L-Q-G

1 μL of CRM197 (32 mg/mL) is added to 30 μL ofCyclooctyne-cyclopropyl-CH₂—OC(O)NH-L-Q-G (2 mg/mL) in 100 mM pH 8 Trisbuffer and 3 μL of microbial transglutaminase (stock of 50 mg/mL in PBS1× prepared from commercial 1% mTGase in maltocyclodextrin) are added.Reaction incubated at 25° C. for 1 hour. One addition of the linker isobserved by Mass Spectrum. LCMS calculated: 58884 observed: (M+1) 58885.

1 μL of CRM197 (32 mg/mL) is added to 30 μL, ofCyclooctyne-cyclopropyl-CH₂—OC(O)NH-L-Q-G (2 mg/mL) in 100 mM pH 8 Trisbuffer and 3 μL of microbial transglutaminase (stock of 50 mg/mL in PBS1× prepared from commercial 1% mTGase in maltocyclodextrin) are added.Reaction incubated at 25° C. for 24 hours. Two additions of the linkerare observed by Mass Spectrum. LCMS calculated: 59360 observed: (M+1)59361.

Z-Q-NH—(CH₂)₃-dimethylacetal

1 μL of CRM197 (32 mg/mL) is added to 50 μL ofZ-Q-NH—(CH₂)₃-dimethylacetal (8 mg/mL) in 100 mM pH 8 Tris buffer and 3uL of microbial transglutaminase (stock of 50 mg/mL in PBS 1× preparedfrom commercial 1% mTGase in maltocyclodextrin) are added. Reactionincubated at 22° C. for 1 hour. One addition of the linker is observedby Mass Spectrum. LCMS calculated: 58787; observed: (M+1) 58788.

Z-Q-NH—(CH₂)₂—NH—C(O)—CH₂-Alexafluor647

100 μL of CRM197 (32 mg/mL) is added to 3000 μL ofZ-Q-NH—(CH₂)₂—NH—C(O)—CH₂-Alexafluor647 (1 mg/mL) in 100 mM pH 8 Trisbuffer and 300 uL of microbial transglutaminase (stock of 50 mg/mL inPBS 1× prepared from commercial 1% mTGase in maltocyclodextrin) areadded. Reaction incubated at 25° C. for 18 hours. Reaction purified viaSEC with a running buffer of PBS 1× over 1.5 CV. One addition of theFluorophore is observed by Mass Spectrum. LCMS calculated: 59554;observed: (M+1) 59556 Yield: 2.6 mg, 81% yield.

Ac-L-Q-G-NH—(CH₂)₂—C(O)—(CH₂)₂—SO₂-Tol

To a solution of Ac-L-Q-G-NH—(CH₂)₂—C(O)—(CH₂)₂—SO₂-Tol (50 μL, 0.084μmol) (roughly 1 mg/mL) was added CRM (0.5 μL, 0.00027 μmol) and TGase(1.5 μL, 1.97E-05 μmol) and mixed at 25° C. for 1 hr. Reaction ˜60%complete. LCMS calculated: 58987 observed: (M+1) 58988. Reaction takendirectly onto further modification with glutathione (procedure below).

Z-Q-NH—(CH₂)₄—C(O)—(CH₂)₂—SO₂-Tol

1 μL of CRM197 (32 mg/mL) is added to 30 μL ofZ-Q-NH—(CH₂)₄—C(O)—(CH₂)₂—SO₂-Tol (1 mg/mL) in 100 mM pH 6 SodiumAcetate buffer and 3 μL of microbial transglutaminase (stock of 50 mg/mLin PBS 1× prepared from commercial 1% mTGase in maltocyclodextrin) areadded. Reaction is incubated at 25° C. for 3 hours. Reaction is purifiedvia a 10 kDa Amicon filter. One addition of the linker was observed byMass Spec. LCMS calculated: 58951 observed: (M+1) 58953.

Z-Q-NH-MenA Polysaccharide

156 μL of CRM197 (32 mg/mL) is added to 5000 μL of Z-Q-NH-MenAPolysaccharide (1 mg/mL) in 100 mM pH 8 Tris buffer and 488 μL ofmicrobial transglutaminase (stock of 50 mg/mL in PBS 1× prepared fromcommercial 1% mTGase in maltocyclodextrin) are added. Reaction incubatedat 25° C. for 18 hours. Reaction purified via 50 kDa Amicon filter whichresulted in a final yield of 2 mg of product (38% yield). Product wasconfirmed by SDS page (in above drawings paragraph 006) as the productis a hetergeneous mixture due to the heterogenity of the polysaccharide.

Z-Q-NH—(CH₂)₅—C(O)-monomethylauristatin F

50 μL of CRM197 (32 mg/mL) is added to 1500 μL ofZ-Q-NH—(CH₂)₅—C(O)-monomethylauristatin F (1 mg/mL) in 100 mM pH 8 Trisbuffer and 150 μL of microbial transglutaminase (stock of 50 mg/mL inPBS 1× prepared from commercial 1% mTGase in maltocyclodextrin) areadded. Reaction incubated at 25° C. for 45 minutes. Reaction purifiedvia SEC with a running buffer of PBS 1× over 1.5 CV. One addition ofMMAF is observed on the Mass Spec. LCMS calculated: 59499 observed:59498 Yield: 659 ug, 38% yield.

60 μL of CRM197 (32 mg/mL) is added to 1500 μL ofZ-Q-NH—(CH₂)₅—C(O)-monomethylauristatin F (1 mg/mL) in 100 mM pH 8 Trisbuffer and 170 μL of microbial transglutaminase (stock of 50 mg/mL inPBS 1× prepared from commercial 1% mTGase in maltocyclodextrin) areadded. Reaction incubated at 25° C. for 24 hours. An additional 60 μL ofmTGase was added, and the reaction was incubated for another 18 hours.Reaction purified via SEC with a running buffer of PBS 1× over 1.5 CV.Two additions of MMAF are observed by the Mass Spectrum. LCMScalculated: 60590 observed: 60589 Yield: 700 ug, 36% yield.

Phenol-(CH2)2-C(O)-L-Q-G

1 μL, of CRM197 (32 mg/mL) is added to 22 μL of Phenol-(CH2)2-C(O)-L-Q-G(0.5 mg/mL) in 100 mM pH 8 Tris buffer and 3 μL of microbialtransglutaminase (stock of 50 mg/mL in PBS 1× prepared from commercial1% mTGase in maltocyclodextrin) are added. Reaction incubated at 25° C.for 1 hour. The addition of one small molecule is observed by Mass Spec.Expected Mass: 58856. Observed Mass: 58857.

Modification of GBS80 with Z-Q-G-NH-(PEG)₃-N₃

2.32 mL GBS80 protein (3.49 mg/mL) was added to 14 mL Z-Q-G-NH-(PEG)₃-N₃(8 mg/mL) in 100 mM sod acetate pH 6 and 50 μL of mTGase (50 mg/ml inPBS) was added. Reaction was incubated overnight at 37° C. LCMS showsaddition of 1 and 2 adducts and a small amount of +3. Reaction wasquenched with 0.8 mL 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicacid (10 mg/mL) and incubated at rt for 1 hr. Reaction was then passedthrough zeba spin column 3×. Recovered material was analyzed by LCMSgiving modified GBS80 in 78% overall yield. LCMS calculated: 53355,53880, 54405; observed: (M+1) 53355, 53877, 54398.

mTGase-Mediated Labelling of CRM197+ZQ(PEG2)Azidobenzylamide

To a solution of ZQ(PEG2)azidobenzylamide (benzyl(17-amino-1-(4-azidophenyl)-2,13,17-trioxo-6,9-dioxa-3,12-diazaheptadecan-14-yl)carbamates)in tris buffer pH 8 (3.5 mg/mL, 86 μL, 0.527 μmol) was added CRM197 (33mg/mL, 7.55 μL, 0.0043 μmol) followed by a solution of transglutaminaseenzyme in PBS (50 mg/mL, 7.61 μL, 0.0100 μmol). The reaction was stirredat 37° C. for 16 hours at which point LCMS analysis showed conversion to+1, +2 and +3 products. LCMS QT2; Protein_(—)35-70 kDa_(—)3 min:R_(t)=1.48 min; MS [M+linker]: observed: 58958, calculated: 58962; MS[M+(2 linkers)]: observed: 59513, calculated: 59514; MS [M+(3 linkers)]:observed: 60067, calculated: 60066.

Degree of Labelling Calculated Observed % R_(t) (min) CRM197 58410 5840819 1.48 CRM197 + 1 ZQ-linker 58962 58958 36 1.48 CRM197 + 2 ZQ-linker59514 59513 25 1.48 CRM197 + 3 ZQ-linker 60066 60067 20 1.48

mTGase-mediated labelling of CRM197+ZQ(PEG)₂amidoethylmethyldiazirin

To a solution of benzyl(18-amino-1-(3-methyl-3H-diazirin-3-yl)-3,14,18-trioxo-7,10-dioxa-4,13-diazaoctadecan-15-yl)carbamatein tris buffer pH 8 (3.5 mg/mL, 50 μL, 0.336 μmol) was added CRM197 (33mg/mL, 4.82 μL, 0.0027 μmol) followed by a solution of transglutaminaseenzyme in PBS (50 mg/mL, 4.85 μL, 0.0064 μmol). The reaction was stirredat r.t. for two days at which point LCMS analysis showed conversion to+1, +2 and +3 product. LCMS QT2; Protein_(—)35-70 kDa_(—)3 min:R_(t)=1.69 min; MS [M+linker]: observed: 58912, calculated: 58913; MS[M+(2× linker)]: observed: 59415, calculated: 59416; MS [M+(3× linker)]:observed: 59918, calculated: 59919.

Degree of Labelling Calculated Observed % R_(t) (min) CRM197 58410 5840811 1.69 CRM197 + 1 ZQ-linker 58913 58912 45 1.69 CRM197 + 2 ZQ-linker59416 59415 32 1.69 CRM197 + 3 ZQ-linker 59919 59918 12 1.69

mTGase-mediated labelling of CRM197+ZQ-FA

To a solution of ZQ-FA in 100 mM tris buffer pH 8 (8 mg/mL, 203 μL,1.316 μmol) was added CRM197 (33 mg/mL, 1.515 μL, 0.00086 μmol) followedby a solution of transglutaminase enzyme in PBS (50 mg/mL, 0.455 μL,0.00060 μmol). The reaction was stirred at r.t. for 16 hours. Thereaction mixture was exchanged into 100 mM tris buffer pH 8 using 10 kDaMWCO Amicon centrifugal filter by diluting and concentrating thereaction 5 times to a volume of 100 pt. LCMS analysis showed conversionto +1, +2, +3 and +4 products. LCMS QT2; Protein_(—)35-70 kDa_(—)3 min:R_(t)=1.45 min; MS [M+ZQ-FA]: observed: 59625, calculated: 59624; MS[M+(2×ZQ-FA)]: observed: 60839, calculated: 60838; MS [M+(3×ZQ-FA)]:observed: 62054, calculated: 62052; MS [M+(4×ZQ-FA)]: observed: 63270,calculated: 63266.

Degree of Labelling Calculated Observed % R_(t) (min) CRM197 58410 n/a 0n/a CRM197 + 1 ZQ-FA 59624 59625 14 1.45 CRM197 + 2 ZQ-FA 60838 60839 231.45 CRM197 + 3 ZQ-FA 62052 62054 35 1.45 CRM197 + 4 ZQ-FA 63266 6327028 1.45

mTGase-Mediated Labelling of CRM197+ Azido Nitrophenyl QG

To a solution of azidonitrophenyl-QG in 100 mM tris buffer pH 8 (8mg/mL, 100 μL) was added CRM197 (33 mg/mL, 1.0 μL, 33 ug) followed by asolution of transglutaminase enzyme in PBS (50 mg/mL, 1.0 μL, 0.1% TGasein maltocyclodextrin). The reaction was incubated at r.t. for 16 hours.LCMS analysis showed conversion to +1, +2, and +3 product. LCMS QT1;Protein_(—)20-70 kDa_(—)3 min: R_(t)=1.67 min; MS [M+1 azido nitrophenylQG]: observed: 58815, calculated: 58803; MS [M+2 azido nitrophenyl QG]:observed: 59191, calculated: 59196; MS [M+3 azido nitrophenyl QG]:observed: 59585, calculated: 59589.

R_(t) Degree of Labelling Calculated Observed % (min) CRM197 58410 584285 n/a CRM197 + 1 58803 58815 50 1.67 azidonitrophenylQG CRM197 + 2 5919659191 40 1.67 azidonitrophenylQG CRM197 + 3 59589 59585 5 1.67azidonitrophenylQG

mTGase-Mediated Labelling of CRM197+ Diazirine-QG

To a solution of diazirine-QG in 100 mM tris buffer pH 8 (8 mg/mL, 100μL) was added CRM197 (33 mg/mL, 1.0 μL, 33 ug) followed by a solution oftransglutaminase enzyme in PBS (50 mg/mL, 1.0 μL, 0.1% TGase inmaltocyclodextrin). The reaction was incubated at r.t. for 2 hours. LCMSanalysis showed conversion to +1 product. LCMS QT1; Protein_(—)20-70kDa_(—)3 min: R_(t)=1.67 min; MS [M+diazirine-QG]: observed: 58705,calculated: 59706.

Degree of Labelling Calculated Observed % R_(t) (min) CRM197 58410 n/a 0n/a CRM197 + 1 diazirine-QG 58706 58705 100 1.67

mTGase-Mediated Labelling of CRM197+ZQ(PEG)₃Biotin

To a solution of ZQ(PEG)₃Biotin in 100 mM tris buffer pH 8 (8 mg/mL, 100μL) was added CRM197 (33 mg/mL, 1.0 μL, 33 ug) followed by a solution oftransglutaminase enzyme in PBS (50 mg/mL, 1.0 μL, 0.1% TGase inmaltocyclodextrin). The reaction was incubated at r.t. for 16 hours.LCMS analysis showed conversion to +1 product. LCMS QT1;Protein_(—)20-70 kDa_(—)3 min: R_(t)=1.67 min; MS [M+1 ZQ(PEG)₃Biotin]:observed: 59073, calculated: 59074; MS [M+2 ZQ(PEG)₃Biotin]: observed:59737, calculated: 59738; MS [M+3 ZQ(PEG)₃Biotin]: observed: 60403,calculated: 60402.

Degree of Labelling Calculated Observed % R_(t) (min) CRM197 58410 none0 n/a CRM197 + 1 ZQ(PEG)₃Biotin 59074 59073 40 1.75 CRM197 + 2ZQ(PEG)₃Biotin 59738 59737 55 1.75 CRM197 + 3 ZQ(PEG)₃Biotin 60402 604035 1.75

Examples of Functionalization of Labeled mTGase Catalyzed SelectiveLysine Labeling of Proteins Z-Q-G-NH-(PEG)₃-N₃

3.2 mg of azido labeled protein was combined with polysaccharide with aconjugation ratio of PS/Prot 6:1 w/w. Product purified by 2×HA column.First run (BLOCK 1=NaPi 2 mM pH 7.2, BLOCK 2=NaPi 400 mM pH 7.2) removesfree protein. Second run (BLOCK 1=NaPi 2 mM/NaCl 550 mM pH 7.2, BLOCK2=NaPi 10 mM pH 7.2, BLOCK 3=NaPi 35 mM pH 7.2, BLOCK 4=NaPi 400 mM pH7.2) removes free polysaccharide.

3.3 mg of azido labeled protein was combined with polysaccharide with aconjugation ratio PS/Prot 6:1 w/w. Product purified by 2×HA column.First run (BLOCK 1=NaPi 2 mM pH 7.2, BLOCK 2=NaPi 400 mM pH 7.2) removesfree protein. Second run (BLOCK 1=NaPi 2 mM/NaCl 550 mM pH 7.2, BLOCK2=NaPi 10 mM pH 7.2, BLOCK 3=NaPi 35 mM pH 7.2, BLOCK 4=NaPi 400 mM pH7.2) removes free polysaccharide.

As shown in FIGS. 2-4, SDS page gel characterization of products ofthese experiments were obtained. The respective yields are also shown inTable 3 below.

TABLE 3 Saccharide/ protein Sacch/ Free used for Yield Conjugation Protsaccharide Protein conjugation (% final Sample Protein chemistry (w/w) %(dionex) TOT mg (w/w) protein) GBS PSV(alk)- X CFCC 4.3 6.7 570.7 6:120.6 GBS80(K-N3) GBS PSII(alk)- X CFCC 1.5 <3.3 1685.7 6:1 24.0GBS80(K-N3)

Each of these clicked GBS80 conjugates obtained through the mTGaselabeling method were tested biologically assays discussed below.

ELISA Immuno Assay for Determination of Ig Titers Against GBS II or VPolysaccharide Antigens

IgG titers against GBS polysaccharides II or V in the sera fromimmunized animals were measured as follows.

Microtiter plates (Nunc Maxisorp) were coated with 100 μl of 1.0 μg/mLHSA-adh (Human Serum Albumin-adipic acid dihydrazide) conjugatedpolysaccharides II or V in Phosphate Buffered Saline (PBS). The platewas incubated overnight at room temperature and then washed three timesin washing buffer (0.05% Tween 20 in PBS). After dispensing 250 μl ofPBS, 2% BSA, 0.05% Tween 20 per well, plates were incubated 90 minutesat 37° C. and then aspirated to remove the post-coating solution. Testsera were diluted 1:400 in PBS, 2% BSA, 0.05% Tween 20. Standard serumwas prepared by pooling hyper immune sera and initial dilutions ofstandard pools were chosen to obtain an optical density (OD) of about2.000 at 405 nm. The plates were incubated for 1 hour at 37° C. and thenwashed with washing buffer and 100 μL of Alkaline Phosphatase-Conjugatedanti-mouse IgG 1:1000 in dilution buffer were dispensed in each well.The plates were incubated 90 minutes at 37° C. and then washed withwashing buffer. 100 μL of a solution of p-NitroPhenylPhosphate (p-NPP)4.0 mg/mL in substrate buffer were dispensed in each well. The plateswere incubated 30 minutes at room temperature and then 100 μL of asolution of EDTA 7% (w/v) disodium salt plus Na₂HPO₄ 3.5% pH 8.0, wereadded to each well to stop the enzymatic reaction. The optical density(OD) at 405 nm was measured. Total IgG titres against GBS polysaccharideantigens (II or V) were calculated by using the Reference Line AssayMethod and results were expressed as arbitrary ELISA Units/mL (EU/mL).For each of the three antigens, the standard serum IgG titer wasarbitrarily assigned a value of 1.0 EU/mL. The IgG titer of each serumwas estimated by interpolating the obtained ODs with the titration curve(bias and slope) of the standard pool. Results are displayed in FIGS. 5and 6.

Mouse Active Maternal Immunization Model

Groups of eight CD-1 female mice (age, 6-8 weeks) were immunized on days1, 21, and 35 with 20 mg of antigen or buffer (PBS) formulated in alumadjuvant. Mice were then mated, and their offspring were challengedintraperitoneally with a GBS dose calculated to induce dead in 90% ofthe pups. Protection values were calculated as [(% dead in control−%dead in vaccine)/% dead in control]×100. Mice were monitored on a dailybasis and killed when they exhibited defined humane endpoints that hadbeen pre-established for the study in agreement with Novartis AnimalWelfare Policies. Statistical analysis was performed using Fisher'sexact test. Results are displayed in Tables 4 and 5 below.

TABLE 4 Antigens Protected\Treated % Protection PBS 18/60 30 CRM-II32/50 64 TT-II 19/30 63 GBS80-II 37/70 53 GBS59-1523-II 59/70 84GBS80-K-N3/PSII 58/69 84 challenge strain type II 5401

TABLE 5 Antigens Protected\Treated % Protection PBS 19/40 47 CRM-V 61/7087 TT-V — — GBS80-V 54/57 95 GBS59-1523-V 69/79 87 GBS80-K- 53/60 88N3/PSV challenge strain type V CJB111

Opsonophagocytosis Assay

The opsonophagocytosis assay was performed using GBS strains as targetcells and HL-60 cell line (ATCC; CCL-240), differentiated intogranulocyte-like cells, by adding 100 mM N, N dimethylformamide (Sigma)to the growth medium for 4 d. Mid-exponential bacterial cells wereincubated at 37° C. for 1 h in the presence of phagocytic cells, 10%baby rabbit complement (Cedarlane), and heat-inactivated mouse antisera.Negative controls consisted of reactions either with preimmune sera, orwithout HL-60, or with heat-inactivated complement. The amount ofopsonophagocytic killing was determined by subtracting the log of thenumber of colonies surviving the 1-h assay from the log of the number ofCFU at the zero time point.

Results of the experiments are shown in FIG. 7. GBS80-K-N₃/PSII OPKA andIgG titers are statistically comparable to GBS80-II conjugate made byrandom K conjugation. OPKA and IgG titers show good correlation with %of survival in challenge animal model.

Ac-L-Q-G-NH—(CH₂)₂—C(O)—(CH₂)₂—SO₂-Tol

L-Glutathione (5 μL, 0.813 umol) was added followed by 50 μL of 250 mMTris HCl buffer pH 8, raising the reaction pH to 8. After 4 hours, allof CRM was labeled with one linker as confirmed by mass spectrometrycharacterization. After another 16 hours at 25° C., all of CRM waslabeled with L-Glutathione. Addition of the L-Glutathione: ExpectedMass: 59138, Observed Mass: 59139.

Peptide Mapping Experimental Summary:

Peptide Mapping Digestion: 5 μg modified CRM197 and positive controlCRM197 samples were reduced with 20 mM DTT and digested with 1/30 (w/w)enzyme/protein at 26° C. overnight with trypsin. An aliquot of trypsindigested protein was further digested with GluC enzyme at 1/20enzyme/protein ratio for 4 hr at 26° C.; note all enzymes purchased fromRoche Diagnostics (Gmbh, Germany).

Reverse Phase LC-MS/MS Analysis: Resulting digested peptides wereanalyzed by liquid chromatography electrospray tandem mass spectrometry(LC-ESI MS/MS) on a Thermo LTQ Orbitrap Discovery (Thermo FisherScientific Inc., Waltham, Mass.) coupled to Agilent CapLC (Santa Clara,Calif.). Loaded ˜10-15 pmole of CRM control and modified CRM197 digestson column at 40° C. (Waters Acuity BEH C18, 1.7 μm, 1×100 mm column) Ran80 min total gradient at 10 μL/min stating at 0-1 min, 4% B, increasedto 7% B at 1.1 min, 45% B at 55 min, then 95% B at 63 min, followed bywashing and column equilibration. Mass spectrometer parameters includeda full scan event using the FTMS analyzer at 30000 resolution from m/z300-2000 for 30 ms. Collision Induced Dissociation MS/MS was conductedon the top seven intense ions (excluding 1+ ions) in the ion trapanalyzer, activated at 500 (for all events) signal intensity thresholdcounts for 30 ms.

Data Analysis and Database Searching: All mass spectra were processed inQual Browser V 2.0.7 (Thermo Scientific). Mascot generic files (mgf)were generated with MS DeconTools (R.D. Smith Lab, PPNL) and searchedusing Mascot V2.3.01 (Matrix Science Inc., Boston, Mass.) databasesearch against the provided protein sequence added to an in-house customdatabase and the SwissProt database (V57 with 513,877 sequences) forcontaminating proteins. Search parameters included: enzyme: semitrypsinor trypsin/Glu-C, allowed up to three missed cleavage; variablemodifications: added expected masses of small molecules (362.147787 Daand 463.206698 Da) to database called “CRM Tgase+alkyne 362 Da mod(CKR), CRM Tgase+alkyne 362 Da mod (N-term), CRM Tgase+azide 463 Da mod(CKR), CRM Tgase+azide 463 Da mod (N-term)”; peptide tolerance: ±20 ppm;MS/MS tolerance: ±0.6 Da. Sequence coverage and small moleculemodification assessments were done on ions scores with >95% confidence.High-scoring peptide ions were then selected for manual MS/MS analysisusing Qual Browser.

Results for CRM+Cyclooctyne-Cyclopropyl-CH₂—OC(O)NH-Q-G

Trypsin digest: 83% sequence coverage; No modification detected at thision score threshold. Trypsin/GluC digest: 97% sequence coverage;Modification detected on Lys37 or Lys39.

CRM Exp095 Trypsin/GluC DigestionSequence Coverage: 91%, Matched peptides shown inBold Text K37 or K39 Modified with CRM Tgase + azide 463 Da mod (CKR)SEQ ID NO: 1 1 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQ

P

SGTQG NYDDDW 51 KEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKV DNAE 101TIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSS VEYI 151NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVG SSLS 201CINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQY LEEF 251HQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADN LEKT 301TAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPL VGEL 351VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAV SWNT 401VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKS KTHI 451SVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSS EKIH 501SNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

Results for CRM+ZQ-NH-(PEG)₃N₃

Trypsin digest: 69% sequence coverage; No modification detected at thision score threshold. Trypsin/GluC digest: 91% sequence coverage;Modification detected on Lys37 or Lys39.

CRM Exp083 Trypsin/GluC DigestionSequence Coverage: 97%, Matched peptides shown inBold Text K37 or K39 Modified with CRM Tgase + alkyne 362 Da mod (CKR)SEQ ID NO: 2 1 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQ

P

SGTQG NYDDDW 51 KEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKV DNAE 101TIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSS VEYI 151NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVG SSLS 201CINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQY LEEF 251HQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADN LEKT 301TAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPL VGEL 351VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAV SWNT 401VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKS KTHI 451SVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSS EKIH 501SNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

CRM Control

Trypsin digest: 85% sequence coverage.

Trypsin/GluC digest: 79% sequence coverage.

It is understood that the invention is not limited to the embodimentsset forth herein for illustration, but embraces all such forms thereofas come within the scope of the above disclosure.

Prophetic Examples Benzyl(17-amino-1-(4-azido2-nitrophenyl)-2,13,17-trioxo-6,9-dioxa-3,12-diazaheptadecan-14-yl)carbamate

4-azido-2-nitrophenylacetic acid N-succinimido ester (0.073 mmol) isdissolved in DMF (1 mL) and combined with a solution of benzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate(20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121 mL, 0.585 mmol) isadded and the reaction is mixed at r.t. for 4 hours. The solution ispurified via MS-triggered HPLC (100-Prep3; Acid_Method 3; Sunfire 30×50mm Sum column ACN/H₂O w/0.1% TFA 75 ml/min, 1.5 ml injection; TubeTrigger M=570). Fractions with desired product are pooled andlyophilized.

ZQ-(PEG2) phenyl trifluoromethyldiazirine

4-trifluoromethyl diazirine phenylacetic acid N-succinimido ester (0.073mmol) is dissolved in DMF (1 mL) and combined with a solution of benzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate(20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121 mL, 0.585 mmol) isadded and the reaction is mixed at r.t. for 4 hours. The solution ispurified via MS-triggered HPLC (100-Prep3; Acid_Method 3; Sunfire 30×50mm Sum column ACN/H₂O w/0.1% TFA 75 ml/min, 1.5 ml injection; TubeTrigger M=570). Fractions with desired product are pooled andlyophilized.

ZQ-(PEG2)-tetrazine

Diazirine N-succinimido ester (0.073 mmol) is dissolved in DMF (1 mL)and combined with a solution of benzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate (20 mg, 0.049 mmol) in DMF(2.3 mL). DIPEA (0.121 mL, 0.585 mmol) is added and the reaction ismixed at r.t. for 4 hours. The solution is purified via MS-triggeredHPLC (100-Prep3; Acid_Method 3; Sunfire 30×50 mm Sum column ACN/H₂Ow/0.1% TFA 75 ml/min, 1.5 ml injection; Tube Trigger M=570). Fractionswith desired product are pooled and lyophilized.

ZQ-(PEG2)-tetrazine

Tetrazine (PEG)N-succinimido ester (0.073 mmol) is dissolved in DMF (1mL) and combined with a solution of benzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate(20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121 mL, 0.585 mmol) isadded and the reaction is mixed at r.t. for 4 hours. The solution ispurified via MS-triggered HPLC (100-Prep3; Acid_Method 3; Sunfire 30×50mm Sum column ACN/H₂O w/0.1% TFA 75 ml/min, 1.5 ml injection; TubeTrigger M=570). Fractions with desired product are pooled andlyophilized.

ZQ(PEG2)-(3aR,4S,7R)-3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran-1,3-dione

(3aR,4S,7R)-3a,4,7,7a-Tetrahydro-4,7-methanoisobenzofuran-1,3-dione(0.162 mmol) (0.073 mmol) is dissolved in DMF (1 mL) and combined with asolution of benzyl(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl)carbamate(0.049 mmol) in DMF (2.3 mL). DIPEA (0.585 mmol) is added and thereaction is mixed at r.t. for 4 hours. The solution is purified viaMS-triggered HPLC (100-Prep3; Acid_Method 3; Sunfire 30×50 mm Sum columnACN/H₂O w/0.1% TFA 75 ml/min, 1.5 ml injection; Tube Trigger M=570).Fractions with desired product are pooled and lyophilized.

Tetrazine-QG

QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL, Ratio: 1.000)and NHS tetrazine (0.148 mmol) is added in H₂O (Volume: 1.000 mL, Ratio:1.000) followed by addition of DIPEA (0.177 mmol). The reaction stirsfor 16 hours at which time the product is purified by HPLC (Sunfire30×50 mm 5 um column ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection) togive the desired product. Fractions are pooled and lyophilized.

Tetrazine(PEG)-QG

QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL, Ratio: 1.000)and NHS PEG_(n), tetrazine (0.148 mmol) is added in H₂O (Volume: 1.000mL, Ratio: 1.000) followed by addition of DIPEA (0.177 mmol). Thereaction stirs for 16 hours at which time the product is purified byHPLC (Sunfire 30×50 mm 5 um column ACN/H2O w/0.1% TFA 75 ml/min 1.5 mlinjection) to give the desired product. Fractions are pooled andlyophilized.

QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL, Ratio: 1.000)and NHS tetrazine (0.148 mmol) is added in H₂O (Volume: 1.000 mL, Ratio:1.000) followed by addition of DIPEA (0.177 mmol). The reaction stirsfor 16 hours at which time the product is purified by HPLC (Sunfire30×50 mm Sum column ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection) togive the desired product. Fractions are pooled and lyophilized.

4-Azido-phenyl-glutamine-glycine

QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL, Ratio: 1.000)and 4-azido-phenylacetic acid N-succinimido ester (0.148 mmol) is addedin H₂O (Volume: 1.000 mL, Ratio: 1.000) followed by addition of DIPEA(0.177 mmol). The reaction stirs for 16 hours at which time the productis purified by HPLC (Sunfire 30×50 mm Sum column ACN/H2O w/0.1% TFA 75ml/min 1.5 ml injection) to give the desired product. Desired fractionsare pooled and lyophilized.

Trifluoromethyldiazirine-benzyl-glutamine-glycine

QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL, Ratio: 1.000)and 4-trifluoromethyl-diazirine-phenylacetic acid N-succinimido ester(0.148 mmol) is added in H₂O (Volume: 1.000 mL, Ratio: 1.000) followedby addition of DIPEA (0.177 mmol). The reaction stirs for 16 hours atwhich time the product is purified by HPLC (Sunfire 30×50 mm Sum columnACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection) to give the desiredproduct. Desired fractions are pooled and lyophilized.

General Procedure for mTGase-Mediated Labelling of CRM197

To a solution of linker in tris buffer pH 8 (3.5 mg/mL, 0.527 μmol orother amount and relative micromolar concentration depending on thelinker identified above) is added CRM197 (33 mg/mL, 7.55 μL, 0.0043μmol) followed by a solution of transglutaminase enzyme in PBS (50mg/mL, 7.61 μL, 0.0100 μmol). The reaction is stirred at rt or 37° C.for 16 hours.

Having thus described exemplary embodiments of the present invention, itshould be noted by those of ordinary skill in the art that the withindisclosures are exemplary only and that various other alternatives,adaptations, and modifications may be made within the scope of thepresent invention. Accordingly, the present invention is not limited tothe specific embodiments as illustrated therein.

1. A method for modifying a protein, comprising: providing a targetprotein having at least one lysine residue; contacting the targetprotein with a modifying compound having the formulaR¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²), in the presence of a microbialtransglutaminase to form a modified protein; wherein x is 0 or 1; y is 0or 1; z is 0 or 1; R¹ is selected from the group consisting of: acetyl,

wherein R⁴ is selected from —H, —N₃, and

W is selected from: C₁—O₆ linear or branched alkyl or polyethyleneglycol having a molecular weight of between about 40 and about 80,000amu; A is absent or selected from —O—, —NH—, and —S—; B is absent orselected from —O—, —C(O)—, —NH—, —C(O)NH—, —NHC(O)—, —NHC(O)O—,—OC(O)NH—, —OC(O)O—, —C═N(OH)—, —S(O₂)—, —NHS(O₂)—, —S(O₂)NH—, —S(O)—,—NHS(O)—, —S(O)NH—; —C(O)O—, —OC(O)—, —S—, ═NH—O—, ═NH—NH— and═NH—N(C₁-C₂₀alkyl)-; R² is selected from the group consisting of: afatty acid, linear or branched C₁-C₃ alkyl-N₃, cyclooctynyl,fluorophore, polysaccharide, —CH(OCH₃)₂,

each n is an integer independently selected from 0 to 6; each Q isselected from H and NO₂. 2-95. (canceled)
 96. A compound of the formulaR¹-(Leu)_(x)-Gln-(Gly)_(y)-(A-W—B—R²)_(z) wherein x is 0 or 1; y is 0 or1; z is 0 or 1; R¹ is selected from the group consisting of: acetyl,

wherein R⁴ is selected from —H, —N₃, and

W is selected from: C₁-C₆ linear or branched alkyl or polyethyleneglycol having a molecular weight of between about 40 and about 80,000amu; A is absent or selected from —O—, —NH—, and —S—; B is absent orselected from —O—, —C(O)—, —NH—, —C(O)NH—, —NHC(O)—, —NHC(O)O—,—OC(O)NH—, —OC(O)O—, —C═N(OH)—, —S(O₂)—, —NHS(O₂)—, —S(O₂)NH—, —S(O)—,—NHS(O)—, —S(O)NH—; —C(O)O—, —OC(O)—, —S—, ═NH—O—, ═NH—NH— and═NH—N(C₁-C₂₀alkyl)-; R² is selected from the group consisting of: afatty acid, linear or branched C₁-C₃ alkyl-N₃, cyclooctynyl,fluorophore, polysaccharide, —CH(OCH₃)₂,

each n is an integer independently selected from 0 to 6; and each Q isselected from H and —NO₂.
 97. The compound of claim 96 selected from thegroup consisting of:


98. A conjugate of a compound of claim
 96. 99. The conjugate claim 98,wherein the conjugate protein is CRM₁₉₇.
 100. The conjugate of claim 98,wherein the conjugate protein is GBS₈₀.
 101. A vaccine comprising aconjugate of claim
 98. 102. A conjugate of a compound of claim
 97. 103.A vaccine comprising a conjugate of claim
 99. 104. A vaccine comprisinga conjugate of claim
 100. 105. A therapeutic protein comprising acompound of claim
 96. 106. A therapeutic protein comprising a compoundof claim
 97. 107. An imaging agent comprising a compound of claim 96.108. An imaging agent comprising a compound of claim
 97. 109. A labelingtool comprising a compound of claim
 96. 110. A labeling tool comprisinga compound of claim 97.